Dosages for treatment with anti-ErbB2 antibodies

ABSTRACT

The present invention concerns the treatment of disorders characterized by the overexpression of ErbB2. More specifically, the invention concerns the treatment of human patients susceptible to or diagnosed with cancer overexpressing ErbB2 with anti-ErbB2 antibody.

RELATED APPLICATIONS

[0001] This application is divisional of U.S. Ser. No. 09/648,067 filedAug. 25, 2000, which claims priority under 35 USC 119(e) to provisionalapplication Nos. 60/151,018, filed Aug. 27, 1999 and 60/213,822, filedJun. 23, 2000, the contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention concerns the treatment of disorderscharacterized by the overexpression of ErbB2 or disorders expressingepidermal growth factor receptor (EGFR), comprising administering to ahuman or animal presenting the disorders a therapeutically effectiveamount of an antibody that binds ErbB2. More specifically, the inventionconcerns the treatment of human patients susceptible to or diagnosedwith cancer overexpressing ErbB2 or expressing EGFR, where the treatmentis with an anti-ErbB2 antibody administered by front loading the dose ofantibody during treatment by intravenous and/or subcutaneousadministration. The invention optionally includes treatment of cancer ina human patient with a combination of an anti-ErbB2 antibody and achemotherapeutic agent, such as, but not limited to, a taxoid. Thetaxoid may be, but is not limited to paclitaxel or docetaxel. Theinvention further includes treatment of cancer in a human patient with acombination of anti-ErbB2 antibody and a chemotherapeutic agent, suchas, but not limited to, an anthracycline derivative. Optionally,treatment with a combination of anti-ErbB2 and an anthracyclinederivative includes treatment with an effective amount of acardioprotectant. The present invention further concerns infrequentdosing of anti-ErbB2 antibodies.

BACKGROUND OF THE INVENTION

[0003] Proto-oncogenes that encode growth factors and growth factorreceptors have been identified to play important roles in thepathogenesis of various human malignancies, including breast cancer. Ithas been found that the human ErbB2 gene (erbB2, also known as her2, orc-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor(p185^(HER2)) related to the epidermal growth factor receptor (EGFR), isoverexpressed in about 25% to 30% of human breast cancer (Slamon et al.,Science 235:177-182 [1987]; Slamon et al., Science 244:707-712 [1989]).

[0004] Several lines of evidence support a direct role for ErbB2 in thepathogenesis and clinical aggressiveness of ErbB2-overexpressing tumors.The introduction of ErbB2 into non-neoplastic cells has been shown tocause their malignant transformation (Hudziak et al., Proc. Natl. Acad.Sci. USA 84:7159-7163 [1987]; DiFiore et al., Science 237:78-182[1987]). Transgenic mice that express HER2 were found to develop mammarytumors (Guy et al., Proc. Natl. Acad. Sci. USA 89:10578-10582 [1992]).

[0005] Antibodies directed against human erbB2 protein products andproteins encoded by the rat equivalent of the erbB2 gene (neu) have beendescribed. Drebin et al., Cell 41:695-706 (1985) refer to an IgG2amonoclonal antibody which is directed against the rat neu gene product.This antibody called 7.16.4 causes down-modulation of cell surface p185expression on B104-1-1 cells (NIH-3T3 cells transfected with the neuproto-oncogene) a inhibits colony formation of these cells. In Drebin etal. PNAS (USA) 83:9129-9133 (1986), the 7.16.4 antibody was shown toinhibit the tumorigenic growth of neu-transformed NIH-3T3 cells as wellas rat neuroblastoma cells (from which the neu oncogene was initiallyisolated) implanted into nude mice. Drebin et al. in Oncogene 2:387-394(1988) discuss the production of a panel of antibodies against the ratneu gene product. Al of the antibodies were found to exert a cytostaticeffect on the growth of neu-transformed cells suspended in soft agar.Antibodies of the IgM, IgG2a and IgG2b isotypes were able to mediatesignificant in vitro lysis of neu-transformed cells in the presence ofcomplement, whereas none of the antibodies were able to mediate highlevels of antibody-dependent cellular cytotoxicity (ADCC) of theneu-transformed cells. Drebin et al. Oncogene 2:273-277 (1988) reportthat mixtures of antibodies reactive with two distinct regions on thep185 molecule result in synergistic anti-tumor effects onneu-transformed NIH-3T3 cells implanted into nude mice. Biologicaleffects of anti-neu antibodies are reviewed in Myers et al., Meth.Enzym. 198:277-290 (1991). See also WO94/22478 published Oct. 13, 1994.Hudziak et al., Mol. Cell. Biol. 9(3): 1165-1172 (1989) describe thegeneration of a panel of anti-ErbB2 antibodies which were characterizedusing the human breast tumor cell line SKBR3. Relative cellproliferation of the SKBR3 cells following exposure to the antibodieswas determined by crystal violet staining of the monolayers after 72hours. Using this assay, maximum inhibition was obtained with theantibody called 4D5 which inhibited cellular proliferation by 56%.Otherantibodies inthepanel, including 7C2 and 7F3, reduced cellularproliferation to a lesser extent in this assay. Hudziak et al. concludethat the effect of the 4D5 antibody on SKBR3 cells was cytostatic ratherthan cytotoxic, since SKBR3 cells resumed growth at a nearly normal ratefollowing removal of the antibody from the medium. The antibody 4D5 wasfurther found to sensitize p185^(erbB2)-overexpressing breast tumor celllines to the cytotoxic effects of TNF-α. See also WO89/06692 publishedJul. 27, 1989. The anti-ErbB2 antibodies discussed in Hudziak et al. arefurther characterized in Fendly et al. Cancer Research 50:1550-1558(1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. GrowthRegulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol.11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986(1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993);Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. CancerResearch 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem.269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5(1991); and D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994).

[0006] Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe twoantibodies which were selected for their reactivity on the lungadenocarcinoma cell line (Calu-3) which overexpresses ErbB2. One of theantibodies, called MGR3, was found to internalize, inducephosphorylation of ErbB2, and inhibit tumor cell growth in vitro.

[0007] McKenzie et al. Oncogene 4:543-548 (1989) generated a panel ofanti-ErbB2 antibodies with varying epitope specificities, including theantibody designated TA1. This TA1 antibody was found to induceaccelerated endocytosis of ErbB2 (see Maier et al. Cancer Res.51:5361-5369 [1991]). Bacus et al. Molecular Carcinogenesis 3:350-362(1990) reported that the TA1 antibody induced maturation of the breastcancer cell lines AU-565 (which overexpresses the erbB2 gene) and MCF-7(which does not). Inhibition of growth and acquisition of a maturephenotype in these cells was found to be associated with reduced levelsof ErbB2 receptor at the cell surface and transient increased levels inthe cytoplasm.

[0008] Stancovski et al. PNAS (USA) 88:8691-8695 (1991) generated apanel of anti-ErbB2 antibodies, injected them i.p. into nude mice andevaluated their effect on tumor growth of murine fibroblasts transformedby overexpression of the erbB2 gene. Various levels of tumor inhibitionwere detected for four of the antibodies, but one of the antibodies(N28) consistently stimulated tumor growth. Monoclonal antibody N28induced significant phosphorylation of the ErbB2 receptor, whereas theother four antibodies generally displayed low or nophosphorylation-inducing activity. The effect of the anti-ErbB2antibodies on proliferation of SKBR3 cells was also assessed. In thisSKBR3 cell proliferation assay, two of the antibodies (N12 and N29)caused a reduction in cell proliferation relative to control. Theability of the various antibodies to induce cell lysis in vitro viacomplement-dependent cytotoxicity (CDC) and antibody-mediatedcell-dependent cytotoxicity (ADCC) was assessed, with the authors ofthis paper concluding that the inhibitory function of the antibodies wasnot attributed significantly to CDC or ADCC.

[0009] Bacus et al. Cancer Research 52:2580-2589 (1992) furthercharacterized the antibodies described in Bacus et al. (1990) andStancovski et al. of the preceding paragraphs. Extending the i.p.studies of Stancovski et al., the effect of the antibodies after i.v.injection into nude mice harboring mouse fibroblasts overexpressinghuman ErbB2 was assessed. As observed in their earlier work, N28accelerated tumor growth, whereas N12 and N29 significantly inhibitedgrowth of the ErbB2-expressing cells. Partial tumor inhibition was alsoobserved with the N24 antibody. Bacus et al. also tested the ability ofthe antibodies to promote a mature phenotype in the human breast cancercell lines AU-565 and MDA-MB453 (which overexpress ErbB2) as well asMCF-7 (containing low levels of the receptor). Bacus et al. saw acorrelation between tumor inhibition in vivo and cellulardifferentiation; the tumor-stimulatory antibody N28 had no effect ondifferentiation, and the tumor inhibitory action of the N12, N29 and N24antibodies correlated with the extent of differentiation they induced.

[0010] Xu et al. Int. J. Cancer 53:401-408 (1993) evaluated a panel ofanti-ErbB2 antibodies for their epitope binding specificities, as wellas their ability to inhibit anchorage-independent andanchorage-dependent growth of SKBR3 cells (by individual antibodies andin combinations), modulate cell-surface ErbB2, and inhibit ligandstimulated anchorage-independent growth. See also WO94/00136 publishedJan. 6, 1994 and Kasprzyk et al. Cancer Research 52:2771-2776 (1992)concerning anti-ErbB2 antibody combinations. Other anti-ErbB2 antibodiesare discussed in Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawveret al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. Cancer Res.54:3758-3765 (1994); and Harwerth et al. J. Biol. Chem. 267:15160-15167(1992).

[0011] A recombinant humanized anti-ErbB2 monoclonal antibody (ahumanized version of the murine anti-ErbB2 antibody 4D5, referred to asrhuMAb HER2, HERCEPTIN®, or HERCEPTIN® anti-ErbB2 antibody) has beenclinically active in patients with ErbB2-overexpressing metastaticbreast cancers that had received extensive prior anti-cancer therapy(Baselga et al., J. Clin. Oncol. 14:737-744 [1996]). The recommendedinitial loading dose for HERCEPTIN® is 4 mg/kg administered as a90-minute infusion. The recommended weekly maintenance dose is 2 mg/kgand can be administered as a 30-minute infusion if the initial loadingdose is well tolerated.

[0012] ErbB2 overexpression is commonly regarded as a predictor of apoor prognosis, especially in patients with primary disease thatinvolves axillary lymph nodes (Slamon et al., [1987] and [1989], supra;Ravdin and Chamness, Gene 159:19-27 [1995]; and Hynes and Stern, BiochimBiophys Acta 1198:165-184 [1994]), and has been linked to sensitivityand/or resistance to hormone therapy and chemotherapeutic regimens,including CMF (cyclophosphamide, methotrexate, and fluoruracil) andanthracyclines (Baselga et al., Oncology 11(3 Supp11):43-48 [1997]).However, despite the association of ErbB2 overexpression with poorprognosis, the odds of HER2-positive patients responding clinically totreatment with taxanes were greater than three times those ofHER2-negative patients (Ibid). rhuMab HER2 was shown to enhance theactivity of paclitaxel (TAXOL®) and doxorubicin against breast cancerxenografts in nude mice injected with BT-474 human breast adenocarcinomacells, which express high levels of HER2 (Baselga et al., Breast Cancer,Proceedings of ASCO, Vol. 13, Abstract 53 [1994]).

SUMMARY OF THE INVENTION

[0013] The present invention concerns the discovery that an earlyattainment of an efficacious target trough serum concentration byproviding an initial dose or doses of anti-ErbB2 antibodies followed bysubsequent doses of equal or smaller amounts of antibody (greater frontloading) is more efficacious than conventional treatments. Theefficacious target trough serum concentration is reached in 4 weeks orless, preferably 3 weeks or less, more preferably 2 weeks or less, andmost preferably 1 week or less, including 1 day or less. The targetserum concentration is thereafter maintained by the administration ofmaintenance doses of equal or smaller amounts for the remainder of thetreatment regimen or until suppression of disease symptoms is achieved.

[0014] The invention further concerns a method for the treatment of ahuman patient susceptible to or diagnosed with a disorder characterizedby overexpression of ErbB2 receptor comprising administering atherapeutically effective amount of an anti-ErbB2 antibodysubcutaneously. Preferably, the initial dose (or doses) as well as thesubsequent maintenance dose or doses are administered subcutaneously.Optionally, where the patient's tolerance to the anti-ErbB2 antibody isunknown, the initial dose is administered by intravenous infusion,followed by subcutaneous administration of the maintenance doses if thepatient's tolerance for the antibody is acceptable.

[0015] According to the invention, the method of treatment involvesadministration of an initial dose of anti-ErbB2 antibody of more thanapproximately 4 mg/kg, preferably more than approximately 5 mg/kg. Themaximum initial dose or a subsequent dose does not exceed 50 mg/kg,preferably does not exceed 40 mg/kg, and more preferably does not exceed30 mg/kg. Administration is by intravenous or subcutaneousadministration, preferably intravenous infusion or bolus injection, ormore preferably subcutaneous bolus injection. The initial dose may beone or more administrations of drug sufficient to reach the targettrough serum concentration in 4 weeks or less, preferably 3 weeks orless, more preferably 2 weeks or less, and most preferably 1 week orless, including one day or less.

[0016] According to the invention, the initial dose or doses is/arefollowed by subsequent doses of equal or smaller amounts of antibody atintervals sufficiently close to maintain the trough serum concentrationof antibody at or above an efficacious target level. Preferably, aninitial dose or subsequent dose does not exceed 50 mg/kg, and eachsubsequent dose is at least 0.01 mg/kg. Preferably the amount of drugadministered is sufficient to maintain the target trough serumconcentration such that the interval between administration cycles is atleast one week. Preferably the trough serum concentration does notexceed 2500 μg/ml and does not fall below 0.01 μg/ml during treatment.The front loading drug treatment method of the invention has theadvantage of increased efficacy by reaching a target serum drugconcentration early in treatment. The subcutaneous delivery ofmaintenance doses according to the invention has the advantage of beingconvenient for the patient and health care professionals, reducing timeand costs for drug treatment. Preferably, the initial dose (or the lastdose within an initial dose series) is separated in time from the firstsubsequent dose by 4 weeks or less, preferably 3 weeks or less, morepreferably 3 weeks or less, most preferably 1 week or less.

[0017] In an embodiment of the invention, the initial dose of anti-ErbB2is 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by intravenous orsubcutaneous administration, such as intravenous infusion orsubcutaneous bolus injection. The subsequent maintenance doses are 2mg/kg delivered once per week by intravenous infusion, intravenous bolusinjection, subcutaneous infusion, or subcutaneous bolus injection. Thechoice of delivery method for the initial and maintenance doses is madeaccording to the ability of the animal or human patient to tolerateintroduction of the antibody into the body. Where the antibody iswell-tolerated, the time of infusion may be reduced. The choice ofdelivery method as disclosed for this embodiment applies to all drugdelivery regimens contemplated according to the invention.

[0018] In another embodiment, the invention includes an initial dose of12 mg/kg anti-ErbB2 antibody, followed by subsequent maintenance dosesof 6 mg/kg once per 3 weeks.

[0019] In still another embodiment, the invention includes an initialdose of 8 mg/kg anti-ErbB2 antibody, followed by 6 mg/kg once per 3weeks.

[0020] In yet another embodiment, the invention includes an initial doseof 8 mg/kg anti-ErbB2 antibody, followed by subsequent maintenance dosesof 8 mg/kg once per week or 8 mg/kg once every 2 to 3 weeks.

[0021] In another embodiment, the invention includes initial doses of atleast 1 mg/kg, preferably 4 mg/kg, anti-ErbB2 antibody on each of days1, 2 and 3, followed by subsequent maintenance doses of 6 mg/kg once per3 weeks.

[0022] In another embodiment, the invention includes an initial dose of4 mg/kg anti-ErbB2 antibody, followed by subsequent maintenance dosesof2 mg/kg twice per week, wherein the maintenance doses are separated by3 days.

[0023] In still another embodiment, the invention includes a cycle ofdosing in which delivery of anti-ErbB2 antibody is 2-3 times per weekfor 3 weeks. In one embodiment of the invention, each dose isapproximately 25 mg/kg or less for a human patient, preferablyapproximately 10 mg/kg or less. This 3 week cycle is preferably repeatedas necessary to achieve suppression of disease symptoms.

[0024] In another embodiment, the invention includes a cycle of dosingin which delivery of anti-ErbB2 antibody is daily for 5 days. Accordingto the invention, the cycle is preferably repeated as necessary toachieve suppression of disease symptoms.

[0025] The disorder preferably is a benign or malignant tumorcharacterized by the overexpression of the ErbB2 receptor, e.g. acancer, such as, breast cancer, squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer. The method of the invention mayfurther comprise administration of a chemotherapeutic agent other thanan anthracycline, e.g. doxorubicin or epirubicin. The chemotherapeuticagent preferably is a taxoid, such as TAXOL® (paclitaxel) or a TAXOL®derivative.

[0026] Preferred anti-ErbB2 antibodies bind the extracellular domain ofthe ErbB2 receptor, and preferably bind to the epitope 4D5 or 3H4 withinthe ErbB2 extracellular domain sequence. More preferably, the antibodyis the antibody 4D5, most preferably in a humanized form. Otherpreferred ErbB2-binding antibodies include, but are not limited to,antibodies 7C2, 7F3, and 2C4, preferably in a humanized form.

[0027] The method of the present invention is particularly suitable forthe treatment of breast or ovarian cancer, characterized by theoverexpression of the ErbB2 receptor.

[0028] The present application also provides a method of therapyinvolving infrequent dosing of an anti-ErbB2 antibody. In particular,the invention provides a method for the treatment of cancer (e.g. cancercharacterized by overexpression of the ErbB2 receptor) in a humanpatient comprising administering to the patient a first dose of ananti-ErbB2 antibody followed by at least one subsequent dose of theantibody, wherein the first dose and subsequent dose are separated fromeach other in time by at least about two weeks (e.g. from about twoweeks to about two months), and optionally at least about three weeks(e.g. from about three weeks to about six weeks). For instance, theantibody may be administered about every three weeks, about two to about20 times, e.g. about six times. The first dose and subsequent dose mayeach be from about 2 mg/kg to about 16 mg/kg; e.g. from about 4 mg/kg toabout 12 mg/kg; and optionally from about 6 mg/kg to about 12 mg/kg.Generally, two or more subsequent doses (e.g. from about two to aboutten subsequent doses) of the antibody are administered to the patient,and those subsequent doses are preferably separated from each other intime by at least about two weeks (e.g. from about two weeks to about twomonths), and optionally at least about three weeks (e.g. from aboutthree weeks to about six weeks). The two or more subsequent doses mayeach be from about 2 mg/kg to about 16 mg/kg; or from about 4 mg/kg toabout 12 mg/kg; or from about 6 mg/kg to about 12 mg/kg. The inventionadditionally provides an article of manufacture, comprising a container,a composition within the container comprising an anti-ErbB2 antibody,and a package insert containing instructions to administer the antibodyaccording to such methods.

[0029] The presently described dosing protocols may be applied to otheranti-ErbB antibodies such as anti-epidermal growth factor receptor(EGFR), anti-ErbB3 and anti-ErbB4 antibodies. Thus, the inventionprovides a method for the treatment of cancer in a human patient,comprising administering an effective amount of an anti-ErbB antibody tothe human patient, the method comprising administering to the patient aninitial dose of at least approximately 5 mg/kg of the anti-ErbBantibody; and administering to the patient a plurality of subsequentdoses of the antibody in an amount that is approximately the same orless than the initial dose. Alternatively, or additionally, theinvention pertains to a method for the treatment of cancer in a humanpatient comprising administering to the patient a first dose of ananti-ErbB antibody followed by at least one subsequent dose of theantibody, wherein the first dose and subsequent dose are separated fromeach other in time by at least about two weeks. The inventionadditionally provides an article of manufacture, comprising a container,a composition within the container comprising an anti-ErbB antibody, anda package insert containing instructions to administer the antibodyaccording to such methods.

[0030] In another aspect, the invention concerns an article ofmanufacture, comprising a container, a composition within the containercomprising an anti-ErbB2 antibody, optionally a label on or associatedwith the container that indicates that the composition can be used fortreating a condition characterized by overexpression of ErbB2 receptor,and a package insert containing instructions to avoid the use ofanthracycline-type chemotherapeutics in combination with thecomposition. According to the invention, the package insert furtherincludes instructions to administer the anti-ErbB2 antibody at aninitial dose of 5 mg/kg followed by the same or smaller subsequent doseor doses. In another embodiment of the invention, the package insertfurther includes instructions to administer the anti-ErbB2 antibodysubcutaneously for at least one of the doses, preferably for all of thesubsequent doses following the initial dose, most preferably for alldoses.

[0031] In a further aspect, the invention provides a method of treatingErbB2 expressing cancer in a human patient comprising administering tothe patient effective amounts of an anti-ErbB2 antibody and achemotherapeutic agent. In one embodiment of the invention, thechemotherapeutic agent is a taxoid including, but not limited to,paclitaxel and docetaxel. In another embodiment, the chemotherapeuticagent is an anthracyline derivative including, but not limited to,doxorubicin or epirubicin. In still another embodiment of the invention,treatment with an anti-ErbB2 antibody and an anthracycline derivativefurther includes administration of a cardioprotectant to the patient. Instill another embodiment, an anthracycline derivative is notadministered to the patient with the anti-ErbB2 antibody. One or moreadditional chemotherapeutic agents may also be administered to thepatient. The cancer is preferably characterized by overexpression ofErbB2.

[0032] The invention further provides an article of manufacturecomprising a container, a composition within the container comprising ananti-ErbB2 antibody and a package insert instructing the user of thecomposition to administer the anti-ErbB2 antibody composition and achemotherapeutic agent to a patient. In another embodiment, thechemotherapeutic agent is other than an anthracycline, and is preferablya taxoid, such as TAXOL®. In still another embodiment, thechemotherapeutic agent is an anthracycline, including but not limitedto, doxorubicin or epirubicin. In yet another embodiment, thechemotherapeutic agent is an anthracycline and the package insertfurther instructs the user to administer a cardioprotectant.

[0033] The methods and compositions of the invention comprise ananti-ErbB2 antibody and include a humanized anti-ErbB2 antibody. Thus,the invention further pertains to a composition comprising an antibodythat binds ErbB2 and the use of the antibody for treating ErbB2expressing cancer, e.g., ErbB2 overexpressing cancer, in a human. Theinvention also pertains to the use of the antibody for treating EGFRexpressing cancer. Preferably the antibody is a monoclonal antibody 4D5,e.g., humanized 4D5 (and preferably huMAb4D5-8 (HERCEPTIN® anti-ErbB2antibody); or monoclonal antibody 2C4, e.g., humanized 2C4. The antibodymay be an intact antibody (e.g., an intact IgG₁ antibody) or an antibodyfragment (e.g., a Fab, F(ab′)₂, diabody, and the like). The variablelight chain and variable heavy chain regions of humanized anti-ErbB2antibody 2C4 are shown in FIGS. 5A and 5B.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 shows epitope-mapping of the extracellular domain of ErbB2as determined by truncation mutant analysis and site-directedmutagenesis (Nakamura et al. J. of Virology 67(10):6179-6191 [October1993]; Renz et al. J. Cell Biol. 125(6):1395-1406 [June 1994]). Theanti-proliferative Mabs 4D5 and 3H4 bind adjacent to the transmembranedomain. The various ErbB2-ECD truncations or point mutations wereprepared from cDNA using polymerase chain reaction technology. The ErbB2mutants were expressed as gD fusion proteins in a mammalian expressionplasmid. This expression plasmid uses the cytomegaloviruspromoter/enhancer with SV40 termination and polyadenylation signalslocated downstream of the inserted cDNA. Plasmid DNA was transfectedinto 293S cells. One day following transfection, the cells weremetabolically labeled overnight in methionine and cysteine-free, lowglucose DMEM containing 1% dialyzed fetal bovine serum and 25 μCi eachof ³⁵S methionine and ³⁵S cysteine. Supernatants were harvested eitherthe ErbB2 MAbs or control antibodies were added to the supernatant andincubated 24 hours at 4° C. The complexes were precipitated, applied toa 10-20% Tricine SDS gradient gel and electrophoresed at 100 V. The gelwas electroblotted onto a membrane and analyzed by autoradiography. SEQID NOs: 8 and 9 depict the 3H4 and 4D5 epitopes, respectively.

[0035]FIG. 2 depicts with underlining the amino acid sequence of Domain1 of ErbB2 (SEQ ID NO: 1). Bold amino acids indicate the location of theepitope recognized by MAbs 7C2 and 7F3 as determined by deletionmapping, i.e. the “7C2/7F3 epitope” (SEQ ID NO:2).

[0036]FIG. 3 is a graph of anti-ErbB2 antibody (HERCEPTIN®) trough serumconcentration (μg/ml, mean±SE, dark circles) by week from week 2 throughweek 36 for ErbB2 overexpressing patients treated with HERCEPTIN®anti-ErbB2 antibody at 4 mg/kg initial dose, followed by 2 mg/kg weekly.The number of patients at each time point is represented by “n” (whitesquares).

[0037]FIG. 4A is a linear plot of tumor volume changes over time in micetreated with HERCEPTIN® anti-ErbB2 antibody. FIG. 4B is asemi-logarithmic plot of the same data as in FIG. 4A such that thevariation in tumor volume for the treated animals is observed morereadily.

[0038]FIGS. 5A and 5B depict alignments of the amino acid sequences ofthe variable light (V_(L)) (FIG. 5A) and variable heavy (V_(H)) (FIG.5B) domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 10 and 11,respectively); V_(L) and V_(H) domains of humanized Fab version 574 (SEQID Nos. 12 and 13, respectively), and human V_(L) and V_(H) consensusframeworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III)(SEQ ID Nos. 14 and 15, respectively). Asterisks identify differencesbetween humanized Fab version 574 and murine monoclonal antibody 2C4 orbetween humanized Fab version 574 and the human framework.Complementarity Determining Regions (CDRs) are in brackets. HumanizedFab version 574, with the changes ArgH71Val, AspH73Arg and IleH69Leu,appears to have binding restored to that of the original chimeric 2C4Fab fragment. Additional FR and/or CDR residues, such as L2, L54, L55,L56, H35 and/or H48, may be modified (e.g. substituted asfollows—IleL2Thr; ArgL54Leu; TyrL55Glu; ThrL56Ser; AspH35Ser; andValH48Ile) in order to further refine or enhance binding of thehumanized antibody. Alternatively, or additionally, the humanizedantibody may be affinity matured in order to further improve or refineits affinity and/or other biological activities.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] I. Definitions

[0040] An “ErbB receptor” is a receptor protein tyrosine kinase whichbelongs to the ErbB receptor family and includes EGFR, HER2, ErbB3 andErbB4 receptors as well as TEGFR (U.S. Pat. No. 5,708,156) and othermembers of this family to be identified in the future. The ErbB receptorwill generally comprise an extracellular domain, which may bind an ErbBligand; a lipophilic transmembrane domain; a conserved intracellulartyrosine kinase domain; and a carboxyl-terminal signaling domainharboring several tyrosine residues which can be phosphorylated. TheErbB receptor may be a native sequence ErbB receptor or an amino acidsequence variant thereof. Preferably the ErbB receptor is nativesequence human ErbB receptor.

[0041] The terms “ErbB1”, “epidermal growth factor receptor” and “EGFR”are used interchangeably herein and refer to native sequence EGFR asdisclosed, for example, in Carpenter et al. Ann. Rev. Biochem.56:881-914 (1987), including variants thereof (e.g. a deletion mutantEGFR as in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1 refersto the gene encoding the EGFR protein product. Examples of antibodieswhich bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRLHB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S.Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such aschimerized 225 (C225) and reshaped human 225 (H225) (see, WO 96/40210,Imclone Systems Inc.).

[0042] “ErbB3” and “HER3” refer to the receptor polypeptide asdisclosed, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968 aswell as Kraus et al. PNAS (USA) 86:9193-9197 (1989), including variantsthereof Examples of antibodies which bind HER3 are described in U.S.Pat. No. 5,968,511 (Akita and Sliwkowski), e.g. the 8B8 antibody (ATCCHB 12070) or a humanized variant thereof

[0043] The terms “ErbB4” and “HER4” herein refer to the receptorpolypeptide as disclosed, for example, in EP Pat Appln No 599,274;Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); andPlowman et al., Nature, 366:473-475 (1993), including variants thereofsuch as the HER4 isoforms disclosed in WO 99/19488.

[0044] The terms “HER2”, “ErbB2” “c-Erb-B2” are used interchangeably.Unless indicated otherwise, the terms “ErbB2” “c-Erb-B2” and “HER2” whenused herein refer to the human protein, and “erbB2,” “c-erb-B2,” and“her2” refer to human gene. The human erbB2 gene and ErbB2 protein are,for example, described in Semba et al., PNAS (USA) 82:6497-6501 (1985)and Yamamoto et al. Nature 319:230-234 (1986) (Genebank accession numberX03363). ErbB2 comprises four domains (Domains 1-4).

[0045] The “epitope 4D5” is the region in the extracellular domain ofErbB2 to which the antibody 4D5 (ATCC CRL 10463) binds. This epitope isclose to the transmembrane region of ErbB2. To screen for antibodieswhich bind to the 4D5 epitope, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed (see FIG. 1) to assesswhether the antibody binds to the 4D5 epitope of ErbB2 (i.e. any one ormore residues in the region from about residue 529, e.g. about residue561 to about residue 625, inclusive).

[0046] The “epitope 3H4” is the region in the extracellular domain ofErbB2 to which the antibody 3H4 binds. This epitope is shown in FIG. 1,and includes residues from about 541 to about 599, inclusive, in theamino acid sequence of ErbB2 extracellular domain.

[0047] The “epitope 7C2/7F3” is the region at the N-terminus of theextracellular domain of ErbB2 to which the 7C2 and/or 7F3 antibodies(each deposited with the ATCC, see below) bind. To screen for antibodieswhich bind to the 7C2/7F3 epitope, a routine cross-blocking assay suchas that described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to establish whether theantibody binds to the 7C2/7F3 epitope on ErbB2 (i.e. any one or more ofresidues in the region from about residue 22 to about residue 53 ofErbB2; SEQ ID NO: 2).

[0048] The term “induces cell death” or “capable of inducing cell death”refers to the ability of the antibody to make a viable cell becomenonviable. The “cell” here is one which expresses the ErbB2 receptor,especially where the cell overexpresses the ErbB2 receptor. A cell which“overexpresses” ErbB2 has significantly higher than normal ErbB2 levelscompared to a noncancerous cell of the same tissue type. Preferably, thecell is a cancer cell, e.g. a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic or bladdercell. In vitro, the cell maybe a SKBR3, BT474, Calu 3, MDA-MB453,MDA-MB-361 or SKOV3 cell. Cell death in vitro may be determined in theabsence of complement and immune effector cells to distinguish celldeath induced by antibody dependent cellular cytotoxicity (ADCC) orcomplement dependent cytotoxicity (CDC). Thus, the assay for cell deathmay be performed using heat inactivated serum (i.e. in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the antibody is able to induce cell death, loss of membraneintegrity as evaluated by uptake of propidium iodide (PI), trypan blue(see Moore et al. Cytotechnology 17:1-11 [1995]) or 7AAD can be assessedrelative to untreated cells. Preferred cell death-inducing antibodiesare those which induce PI uptake in the “PI uptake assay in BT474cells”.

[0049] The phrase “induces apoptosis” or “capable of inducing apoptosis”refers to the ability of the antibody to induce programmed cell death asdetermined by binding of annexin V, fragmentation of DNA, cellshrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/orformation of membrane vesicles (called apoptotic bodies). The cell isone which overexpresses the ErbB2 receptor. Preferably the “cell” is atumor cell, e.g. a breast, ovarian, stomach, endometrial, salivarygland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Invitro, the cell may be a SKBR3, BT474, Calu 3 cell, MDA-MB-453,MDA-MB-361 or SKOV3 cell. Various methods are available for evaluatingthe cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNAfragmentation can be evaluated through DNA laddering as disclosed in theexample herein; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcell in an “annexin binding assay using BT474 cells” (see below).

[0050] Sometimes the pro-apoptotic antibody will be one which blocks HRGbinding/activation of the ErbB2/ErbB3 complex (e.g. 7F3 antibody). Inother situations, the antibody is one which does not significantly blockactivation of the ErbB2/ErbB3 receptor complex by HRG (e.g. 7C2).Further, the antibody may be one like 7C2 which, while inducingapoptosis, does not induce a large reduction in the percent of cells inS phase (e.g. one which only induces about 0-10% reduction in thepercent of these cells relative to control).

[0051] The antibody of interest may be one like 7C2 which bindsspecifically to human ErbB2 and does not significantly cross-react withother proteins such as those encoded by the erbB1, erbB3 and/or erbB4genes. Sometimes, the antibody may not significantly cross-react withthe rat neu protein, e.g., as described in Schecter et al. Nature312:513 (1984) and Drebin et al., Nature 312:545-548 (1984). In suchembodiments, the extent of binding of the antibody to these proteins(e.g., cell surface binding to endogenous receptor) will be less thanabout 10% as determined by fluorescence activated cell sorting (FACS)analysis or radioimmunoprecipitation (RIA).

[0052] “Heregulin” (HRG) when used herein refers to a polypeptide whichactivates the ErbB2-ErbB3 and ErbB2-ErbB4 protein complexes (i.e.induces phosphorylation of tyrosine residues in the complex upon bindingthereto). Various heregulin polypeptides encompassed by this term aredisclosed in Holmes et al., Science, 256:1205-1210 (1992); WO 92/20798;Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994); and Marchionni etal., Nature, 362:312-318 (1993), for example. The term includesbiologically active fragments and/or variants of a naturally occurringHRG polypeptide, such as an EGF-like domain fragment thereof (e.g.HRGβ1₁₇₇-244 ).

[0053] The “ErbB2-ErbB3 protein complex” and “ErbB2-ErbB4 proteincomplex” are noncovalently associated oligomers of the ErbB2 receptorand the ErbB3 receptor or ErbB4 receptor, respectively. The complexesform when a cell expressing both of these receptors is exposed to HRGand can be isolated by immunoprecipitation and analyzed by SDS-PAGE asdescribed in Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665(1994).

[0054] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

[0055] “Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

[0056] The term “variable” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light-chain and theheavy-chain variable domains. The more highly conserved portions ofvariable domains are called the framework region (FR). The variabledomains of native heavy and light chains each comprise four FR regions,largely adopting a β-sheet configuration, connected by three CDRs, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FRs and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,NIH Publ. No.91-3242, Vol. 1, pages 647-669 [1991]). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity.

[0057] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, whose name reflectsits ability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-combining sites and is still capable ofcross-linking antigen.

[0058] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0059] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

[0060] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains.

[0061] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these maybe further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

[0062] The term “antibody” is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

[0063] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0064] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i. e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

[0065] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

[0066] “Humanized” forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementarity determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321:522-525 (1986); Reichmannet al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992). The humanized antibody includes a PRIMATIZED™ antibodywherein the antigen-binding region of the antibody is derived from anantibody produced by immunizing macaque monkeys with the antigen ofinterest.

[0067] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

[0068] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0069] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0070] As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG₁,IgG₂, IgG₃, or IgG₄) that is responsible for increasing the in vivoserum half-life of the IgG molecule.

[0071] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented.

[0072] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal is human.

[0073] A “disorder” is any condition that would benefit from treatmentwith the anti-ErbB2 antibody. This includes chronic and acute disordersor diseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include benign and malignant tumors; leukemias andlymphoid malignancies; neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.

[0074] The term “therapeutically effective amount” is used to refer toan amount having antiproliferative effect. Preferably, thetherapeutically effective amount has apoptotic activity, or is capableof inducing cell death, and preferably death of benign or malignanttumor cells, in particular cancer cells. Efficacy can be measured inconventional ways, depending on the condition to be treated. For cancertherapy, efficacy can, for example, be measured by assessing the time todisease progression (TTP), or determining the response rates (RR) (seeExample 1, below). Therapeutically effective amount also refers to atarget serum concentration, such as a trough serum concentration, thathas been shown to be effective in suppressing disease symptoms whenmaintained for a period of time.

[0075] The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

[0076] The term “cytotoxic agent” as used herein refers to a substancethat inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g. I¹³¹, I¹²⁵, Y⁹⁰ and Re⁸⁶), chemotherapeutic agents, andtoxins such as enzymatically active toxins of bacterial, fungal, plantor animal origin, or fragments thereof

[0077] A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidaniine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C” ); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

[0078] A “growth inhibitory agent” when used herein refers to a compoundor composition which inhibits growth of a cell, especially anErbB2-overexpressing cancer cell either in vitro or in vivo. Thus, thegrowth inhibitory agent is one which significantly reduces thepercentage of ErbB2 overexpressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), TAXOL®, and topo II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders:Philadelphia, 1995), especially p. 13. The 4D5 antibody (and functionalequivalents thereof) can also be employed for this purpose.

[0079] “Doxorubicin” is an athracycline antibiotic. The full chemicalname of doxorubicin is(8S-cis)-10-[(3amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

[0080] The term “cytokine” is a generic term for proteins released byone cell population which act on another cell as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

[0081] The term “prodrug” as used in this application refers to aprecursor or derivative form of a pharmaceutically active substance thatis less cytotoxic to tumor cells compared to the parent drug and iscapable of being enzymatically activated or converted into the moreactive parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy”Biochemical Society Transactions, 14, pp.375-382, 615th Meeting Belfast(1986) and Stella et al., “Prodrugs: A Chemical Approach to TargetedDrug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985). The prodrugs of this invention include,but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

[0082] By “solid phase” is meant a non-aqueous matrix to which theantibodies used in accordance with the present invention can adhere.Examples of solid phases encompassed herein include those formedpartially or entirely of glass (e.g.,controlled pore glass),polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinylalcohol and silicones. In certain embodiments, depending on the context,the solid phase can comprise the well of an assay plate; in others it isa purification column (e.g.,an affinity chromatography column). Thisterm also includes a discontinuous solid phase of discrete particles,such as those described in U.S. Pat. No. 4,275,149.

[0083] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as the anti-ErbB2 antibodies disclosed herein and,optionally, a chemotherapeutic agent) to a mammal. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes.

[0084] The term “package insert” is used to refer to instructionscustomarily included in commercial packages of therapeutic products,that contain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch therapeutic products.

[0085] The term “serum concentration,” “serum drug concentration,” or“serum HERCEPTIN® anti-ErbB2 antibody concentration” refers to theconcentration of a drug, such as HERCEPTIN® anti-ErbB2 antibody, in theblood serum of an animal or human patient being treated with the drug.Serum concentration of HERCEPTIN® anti-ErbB2 antibody, for example, ispreferably determined by immunoassay. Preferably, the immunoassay is anELISA according to the procedure disclosed herein.

[0086] The term “peak serum concentration” refers to the maximal serumdrug concentration shortly after delivery of the drug into the animal orhuman patient, after the drug has been distributed throughout the bloodsystem, but before significant tissue distribution, metabolism orexcretion of drug by the body has occurred.

[0087] The term “trough serum concentration” refers to the serum drugconcentration at a time after delivery of a previous dose andimmediately prior to delivery of the next subsequent dose of drug in aseries of doses. Generally, the trough serum concentration is a minimumsustained efficacious drug concentration in the series of drugadministrations. Also, the trough serum concentration is frequentlytargeted as a minimum serum concentration for efficacy because itrepresents the serum concentration at which another dose of drug is tobe administered as part of the treatment regimen. If the delivery ofdrug is by intravenous administration, the trough serum concentration ismost preferably attained within 1 day of a front loading initial drugdelivery. If the delivery of drug is by subcutaneous administration, thepeak serum concentration is preferably attained in 3 days or less.According to the invention, the trough serum concentration is preferablyattained in 4 weeks or less, preferably 3 weeks or less, more preferably2 weeks or less, most preferably in 1 week or less, including 1 day orless using any of the drug delivery methods disclosed herein.

[0088] The term “intravenous infusion” refers to introduction of a druginto the vein of an animal or human patient over a period of timegreater than approximately 5 minutes, preferably between approximately30 to 90 minutes, although, according to the invention, intravenousinfusion is alternatively administered for 10 hours or less.

[0089] The term “intravenous bolus” or “intravenous push” refers to drugadministration into a vein of an animal or human such that the bodyreceives the drug in approximately 15 minutes or less, preferably 5minutes or less.

[0090] The term “subcutaneous administration” refers to introduction ofa drug under the skin of an animal or human patient, preferable within apocket between the skin and underlying tissue, by relatively slow,sustained delivery from a drug receptacle. The pocket may be created bypinching or drawing the skin up and away from underlying tissue.

[0091] The term “subcutaneous infusion” refers to introduction of a drugunder the skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

[0092] The term “subcutaneous bolus” refers to drug administrationbeneath the skin of an animal or human patient, where bolus drugdelivery is preferably less than approximately 15 minutes, morepreferably less than 5 minutes, and most preferably less than 60seconds. Administration is preferably within a pocket between the skinand underlying tissue, where the pocket is created, for example, bypinching or drawing the skin up and away from underlying tissue.

[0093] The term “front loading” when referring to drug administration ismeant to describe an initially higher dose followed by the same or lowerdoses at intervals. The initial higher dose or doses are meant to morerapidly increase the animal or human patient's serum drug concentrationto an efficacious target serum concentration. According to the presentinvention, front loading is achieved by an initial dose or dosesdelivered over three weeks or less that causes the animal's or patient'sserum concentration to reach a target serum trough concentration.Preferably, the initial front loading dose or series of doses isadministered in two weeks or less, more preferably in 1 week or less,including 1 day or less. Most preferably, where the initial dose is asingle dose and is not followed by a subsequent maintenance dose for atleast 1 week, the initial dose is administered in 1 day or less. Wherethe initial dose is a series of doses, each dose is separated by atleast 3 hours, but not more than 3 weeks or less, preferably 2 weeks orless, more preferably 1 week or less, most preferably 1 day or less. Toavoid adverse immune reaction to an antibody drug such as an anti-ErbB2antibody (e.g., HERCEPTIN® anti-ErbB2 antibody) in an animal or patientwho has not previously been treated with the antibody, it may bepreferable to deliver initial doses of the antibody by intravenousinfusion. The present invention includes front loading drug delivery ofinitial and maintenance doses by infusion or bolus administration,intravenously or subcutaneously.

[0094] Published information related to anti-ErbB2 antibodies includesthe following issued patents and published applications: PCT/US89/00051, published Jan. 5, 1989; PCT/US90/02697, published May 18, 1990; EU0474727 issued Jul. 23, 1997; DE 69031120.6, issued Jul. 23, 1997;PCT/US97/18385, published Oct. 9, 1997; SA 97/9185, issued Oct. 14,1997; U.S. Pat. No. 5,677,171, issued Oct. 14, 1997; U.S. Pat. No.5,720,937, issued Feb. 24, 1998; U.S. Pat. No. 5,720,954, issued Feb.24, 1998; U.S. Pat. No. 5,725,856, issued Mar. 10, 1998; U.S. Pat. No.5,770,195, issued Jun. 23, 1998; U.S. Pat. No. 5,772,997, issued Jun.30, 1998; PCT/US98/2626, published Dec. 10, 1998; and PCT/US99/06673,published Mar. 26, 1999, each of which patents and publications isherein incorporated by reference in its entirety.

[0095] II. Production of anti-ErbB2 Antibodies

[0096] A description follows as to exemplary techniques for theproduction of the antibodies used in accordance with the presentinvention. The ErbB2 antigen to be used for production of antibodies maybe, e.g., a soluble form of the extracellular domain of ErbB2 or aportion thereof, containing the desired epitope. Alternatively, cellsexpressing ErbB2 at their cell surface (e.g. NIH-3T3 cells transformedto overexpress ErbB2; or a carcinoma cell line such as SKBR3 cells, seeStancovski et al., PNAS (USA) 88:8691-8695 [1991]) can be used togenerate antibodies. Other forms of ErbB2 useful for generatingantibodies will be apparent to those skilled in the art. (

[0097] i) Polyclonal Antibodies

[0098] Polyclonal antibodies are preferably raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and an adjuvant. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

[0099] Animals are immunized against the antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg or 5 μg of theprotein or conjugate (for rabbits or mice, respectively) with 3 volumesof Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with ⅕ to{fraction (1/10)} the original amount of peptide or conjugate inFreund's complete adjuvant by subcutaneous injection at multiple sites.Seven to 14 days later the animals are bled and the serum is assayed forantibody titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the sameantigen, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

[0100] (ii) Monoclonal Antibodies

[0101] Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Thus, themodifier “monoclonal” indicates the character of the antibody as notbeing a mixture of discrete antibodies.

[0102] For example, the monoclonal antibodies may be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat.No.4,816,567).

[0103] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103 [Academic Press, 1986]).

[0104] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0105] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 or X63-Ag8-653 cells available from the AmericanType Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 [Marcel Dekker, Inc., New York, 1987]).

[0106] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

[0107] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

[0108] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103[Academic Press, 1986]). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

[0109] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0110] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

[0111] In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 [1992]), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 [1993]). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

[0112] The DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567;Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 [1984]), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

[0113] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

[0114] (iii) Humanized and Human Antibodies

[0115] Methods for humanizing non-human antibodies are well known in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al. Science, 239:1534-1536[1988]), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

[0116] The choice of human variable domains, both light and heavy, to beused in making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901[1987]). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 [1993]).

[0117] It is further important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

[0118] Alternatively, it is now possible to produce transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal. Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 [1991]).

[0119] (iv) Antibody Fragments

[0120] Various techniques have been developed for the production ofantibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992) andBrennan et al., Science, 229:81 [1985]). However, these fragments cannow be produced directly by recombinant host cells. For example, theantibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10: 163-167 [1992]). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner. In otherembodiments, the antibody of choice is a single chain Fv fragment(scFv). See WO 93/16185.

[0121] (v) Bispecific Antibodies

[0122] Bispecific antibodies are antibodies that have bindingspecificities for at least two different epitopes. Exemplary bispecificantibodies may bind to two different epitopes of the ErbB2 protein. Forexample, one arm may bind an epitope in Domain 1 of ErbB2 such as the7C2/7F3 epitope, the other may bind a different ErbB2 epitope, e.g. the4D5 epitope. Other such antibodies may combine an ErbB2 binding sitewith binding site(s) for EGFR, ErbB3 and/or ErbB4. Alternatively, ananti-ErbB2 arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 orCD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII(CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms tothe ErbB2-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express ErbB2. These antibodiespossess an ErbB2-binding arm and an arm which binds the cytotoxic agent(e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

[0123] Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 [1983]). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

[0124] According to a different approach, antibody variable domains withthe desired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

[0125] In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0126] According to another approach described in WO96/2701 1, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

[0127] Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

[0128] Techniques for generating bispecific antibodies from antibodyfragments have also been described in the literature. For example,bispecific antibodies can be prepared using chemical linkage. Brennan etal., Science, 229: 81 (1985) describe a procedure wherein intactantibodies are proteolytically cleaved to generate F(ab′)₂ fragments.These fragments are reduced in the presence of the dithiol complexingagent sodium arsenite to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

[0129] Recent progress has facilitated the direct recovery of Fab′-SHfragments from E. coli, which can be chemically coupled to formbispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992)describe the production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

[0130] Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0131] Antibodies with more than two valencies are contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147: 60 (1991).

[0132] (vi) Screening for Antibodies with the Desired Properties

[0133] Techniques for generating antibodies have been described above.Those antibodies having the characteristics described herein areselected.

[0134] To select for antibodies which induce cell death, loss ofmembrane integrity as indicated by, e.g., PI, trypan blue or 7AAD uptakeis assessed relative to control. The preferred assay is the “PI uptakeassay using BT474 cells”. According to this assay, BT474 cells (whichcan be obtained from the American Type Culture Collection [Rockville,Md.]) are cultured in Dulbecco's Modified Eagle Medium (D-MEM):Ham'sF-12 (50:50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2mM L-glutamine. (Thus, the assay is performed in the absence ofcomplement and immune effector cells). The BT474 cells are seeded at adensity of 3×10⁶ per dish in 100×20 mm dishes and allowed to attachovernight. The medium is then removed and replaced with fresh mediumalone or medium containing 10 μg/ml of the appropriate MAb. The cellsare incubated for a 3 day time period. Following each treatment,monolayers are washed with PBS and detached by trypsinization. Cells arethen centrifuged at 1200 rpm for 5 minutes at 4° C., the pelletresuspended in 3 ml ice cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4,140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake areselected.

[0135] In order to select for antibodies which induce apoptosis, an“annexin binding assay using BT474 cells” is available. The BT474 cellsare cultured and seeded in dishes as discussed in the precedingparagraph. The medium is then removed and replaced with fresh mediumalone or medium containing 10 μg/ml of the MAb. Following a three dayincubation period, monolayers are washed with PBS and detached bytrypsinization. Cells are then centrifuged, resuspended in Ca²⁺ bindingbuffer and aliquoted into tubes as discussed above for the cell deathassay. Tubes then receive labeled annexin (e.g. annexin V-FTIC) (1μg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer andFACSCONVERT™ CellQuest software (Becton Dickinson). Those antibodieswhich induce statistically significant levels of annexin bindingrelative to control are selected as apoptosis-inducing antibodies.

[0136] In addition to the annexin binding assay, a “DNA staining assayusing BT474 cells” is available. In order to perform this assay, BT474cells which have been treated with the antibody of interest as describedin the preceding two paragraphs are incubated with 9 μg/ml HOECHST33342™ for 2 hr at 37° C., then analyzed on an EPICS ELITE™ flowcytometer (Coulter Corporation) using MODFIT LT™ software (VeritySoftware House). Antibodies which induce a change in the percentage ofapoptotic cells which is 2 fold or greater (and preferably 3 fold orgreater) than untreated cells (up to 100% apoptotic cells) may beselected as pro-apoptotic antibodies using this assay.

[0137] To screen for antibodies which bind to an epitope on ErbB2 boundby an antibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed by methods known in theart.

[0138] To identify anti-ErbB2 antibodies which inhibit growth of SKBR3cells in cell culture by 50-100%, the SKBR3 assay described in WO89/06692 can be performed. According to this assay, SKBR3 cells aregrown in a 1:1 mixture of F12 and DMEM medium supplemented with 10%fetal bovine serum, glutamine and penicillinstreptomycin. The SKBR3cells are plated at 20,000 cells in a 35 mm cell culture dish (2 mls/35mm dish). 2.5 μg/ml of the anti-ErbB2 antibody is added per dish. Aftersix days, the number of cells, compared to untreated cells are countedusing an electronic COULTER™ cell counter. Those antibodies whichinhibit growth of the SKBR3 cells by 50-100% are selected forcombination with the apoptotic antibodies as desired.

[0139] (vii) Effector Function Engineering

[0140] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance the effectiveness of theantibody in treating cancer, for example. For example, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff etal. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

[0141] (viii) Immunoconjugates

[0142] The invention also pertains to immunoconjugates comprising theantibody described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

[0143] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-ErbB2 antibodies. Examples include ²¹²Bi, ¹³¹I,¹³In, ⁹⁰Y and ¹⁸⁶Re.

[0144] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifinctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as his(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026.

[0145] In another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g. a radionucleotide).

[0146] (ix) Immunoliposomes

[0147] The anti-ErbB2 antibodies disclosed herein may also be formulatedas immunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0148] Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst.81(19)1484 (1989).

[0149] (x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

[0150] The antibodies of the present invention may also be used in ADEPTby conjugating the antibody to a prodrug-activating enzyme whichconverts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378and U.S. Pat. No. 4,975,278.

[0151] The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way so asto covert it into its more active, cytotoxic form.

[0152] Enzymes that are useful in the method of this invention include,but are not limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature 328:457-458 [1987]). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

[0153] The enzymes of this invention can be covalently bound to theanti-ErbB2 antibodies by techniques well known in the art such as theuse of the heterobifunctional crosslinking reagents discussed above.Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 [1984]).

[0154] (xi) Antibody-Salvage Receptor Binding Epitope Fusions

[0155] In certain embodiments of the invention, it may be desirable touse an antibody fragment, rather than an intact antibody, to increasetumor penetration, for example. In this case, it may be desirable tomodify the antibody fragment in order to increase its serum half life.This may be achieved, for example, by incorporation of a salvagereceptor binding epitope into the antibody fragment (e.g by mutation ofthe appropriate region in the antibody fragment or by incorporating theepitope into a peptide tag that is then fused to the antibody fragmentat either end or in the middle, e.g. by DNA or peptide synthesis).

[0156] A systematic method for preparing such an antibody variant havingan increased in vivo half-life comprises several steps. The firstinvolves identifying the sequence and conformation of a salvage receptorbinding epitope of an Fc region of an IgG molecule. Once this epitope isidentified, the sequence of the antibody of interest is modified toinclude the sequence and conformation of the identified binding epitope.After the sequence is mutated, the antibody variant is tested to see ifit has a longer in vivo half-life than that of the original antibody. Ifthe antibody variant does not have a longer in vivo half-life upontesting, its sequence is further altered to include the sequence andconformation of the identified binding epitope. The altered antibody istested for longer in vivo half-life, and this process is continued untila molecule is obtained that exhibits a longer in vivo half-life.

[0157] The salvage receptor binding epitope being thus incorporated intothe antibody of interest is any suitable such epitope as defined above,and its nature will depend, e.g., on the type of antibody beingmodified. The transfer is made such that the antibody of interest stillpossesses the biological activities described herein.

[0158] The epitope preferably constitutes a region wherein any one ormore amino acid residues from one or two loops of a Fc domain aretransferred to an analogous position of the antibody fragment. Even morepreferably, three or more residues from one or two loops of the Fcdomain are transferred. Still more preferred, the epitope is taken fromthe CH2 domain of the Fc region (e.g., of an IgG) and transferred to theCH₁, CH₃, or V_(H) region, or more than one such region, of theantibody. Alternatively, the epitope is taken from the CH2 domain of theFc region and transferred to the C_(L) region or V_(L) region, or both,of the antibody fragment.

[0159] In one most preferred embodiment, the salvage receptor bindingepitope comprises the sequence (5′ to 3′): PKNSSMISNTP (SEQ ID NO: 3),and optionally further comprises a sequence selected from the groupconsisting of HQSLGTQ (SEQ ID NO: 4), HQNLSDGK (SEQ ID NO: 5), HQNISDGK(SEQ ID NO: 6), or VISSHLGQ (SEQ ID NO: 7), particularly where theantibody fragment is a Fab or F(ab′)₂. In another most preferredembodiment, the salvage receptor binding epitope is a polypeptidecontaining the sequence(s)(5′ to 3′): HQNLSDGK (SEQ ID NO: 5), HQNISDGK(SEQ ID NO: 6), or VISSHLGQ (SEQ ID NO: 7) and the sequence: PKNSSMISNTP(SEQ ID NO: 3).

[0160] (xii) Purification of anti-ErbB2 Antibody

[0161] When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems arepreferably first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

[0162] The antibody composition prepared from the cells can be purifiedusing, for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 [1983]). G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 [1986]). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

[0163] Following any preliminary purification step(s), the mixturecomprising the antibody of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g. from about 0-0.25M salt).

[0164] III. Determination of anti-ErbB2 Antibody Concentration in Serum

[0165] The following non-limiting assay is useful for determining thepresence of and to quantitate the amount of specific rhuMAb HER2(humanized anti-p185^(HER2) monoclonal antibody, including HERCEPTIN®anti-ErbB2 antibody) in a body fluid of a mammal including, but notlimited to, serum, amniotic fluid, milk, umbilical cord serum, ocularaqueous and vitreous liquids, and ocular vitreous gel.

[0166] Plate Binding Activity Assay for rhuMAb HER2 (HumanizedAnti-p185^(HER2) Monoclonal Antibody

[0167] The method of assaying rhuMAb HER2 described herein is meant asan example of such a method and is not meant to be limiting. Astandardized preparation of rhuMAb HER2 (Genentech, Inc., South SanFrancisco, Calif.), controls, and serum samples were diluted with AssayDiluent (PBS/0.5% BSA/0.05% Polysorbate 20/0.01% Thimerosal). Thedilutions of standardized rhuMAb HER2 were prepared to span a range ofconcentrations useful for a standard curve. The samples were diluted tofall within the standard curve.

[0168] An aliquot of Coat Antigen in Coating buffer (recombinantp185^(HER2) (Genentech, Inc.) in 0.05 M sodium carbonate buffer) wasadded to each well of a microtiter plate and incubated at 2-8° C. for12-72 hours. The coating solution was removed and each well was washedsix times with water, then blotted to remove excess water.

[0169] An aliquot of Assay Diluent was added to each well and incubatedfor 1-2 hours at ambient temperature with agitation. The wells werewashed as in the previous step.

[0170] Aliquots of diluted standard, control and sample solutions wereadded to the wells and incubated at ambient temperature for 1 hour withagitation to allow binding of the antibody to the coating antigen. Thewells are washed again with water as in previous steps.

[0171] Horse radish peroxidase-conjugate (HRP-conjugate, Goat anti-humanIgG Fc conjugated to horseradish peroxidase; Organon Teknika catalog#55253 or equivalent) was diluted with Assay Diluent to yield anappropriate optical density range between the highest and loweststandards. An aliquot of the HRP-conjugate solution was added to eachwell and incubated at ambient temperature for I hour with agitation. Thewells were washed with water as in previous steps.

[0172] An aliquot of Substrate Solution (o-phenylenediamine (OPD) 5 mgtablet (Sigma P6912 or equivalent) in 12.5 ml 4 mM H₂O₂ in PBS) wasadded to each well and incubated for a sufficient period of time(approximately 8-10 minutes) in the dark at ambient temperature to allowcolor development. The reaction was stopped with an aliquot of 4.5 Nsulfuric acid. Optical density was read at 490-492 nm for detectionabsorbance and 405 nm for reference absorbance. The standard curve dataare plotted and the results for the controls and samples are determinedfrom the standard curve.

[0173] IV. Pharmaceutical Formulations

[0174] Therapeutic formulations of the antibodies used in accordancewith the present invention are prepared for storage by mixing anantibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed.[1980]), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Preferred lyophilized anti-ErbB2 antibody formulations are described inWO 97/04801, expressly incorporated herein be reference.

[0175] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. For example, it may be desirable to further provideantibodies which bind to EGFR, ErbB2 (e.g. an antibody which binds adifferent epitope on ErbB2), ErbB3, ErbB4, or vascular endothelialgrowth factor (VEGF) in the one formulation. Alternatively, or inaddition, the composition may comprise a cytotoxic agent, cytokine orgrowth inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

[0176] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed. (1980).

[0177] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0178] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g. films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S-S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0179] V. Treatment with the Anti-ErbB2 Antibodies

[0180] It is contemplated that, according to the present invention, theanti-ErbB2 antibodies may be used to treat various conditionscharacterized by overexpression and/or activation of the ErbB2 receptor.Exemplary conditions or disorders include benign or malignant tumors(e.g. renal, liver, kidney, bladder, breast, gastric, ovarian,colorectal, prostate, pancreatic, lung, vulval, thyroid, hepaticcarcinomas; sarcomas; glioblastomas; and various head and neck tumors);leukemias and lymphoid malignancies; other disorders such as neuronal,glial, astrocytal, hypothalamic and other glandular, macrophagal,epithelial, stromal and blastocoelic disorders; and inflammatory,angiogenic and immunologic disorders.

[0181] The antibodies of the invention are administered to a humanpatient, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

[0182] The treatment of the present invention involves theadministration of an anti-ErbB2 antibody to an animal or human patient,followed at intervals by subsequent doses of equal or smaller doses suchthat a target serum concentration is achieved and maintained duringtreatment. Preferably, maintenance doses are delivered by bolusdelivery, preferably by subcutaneous bolus administration, makingtreatment convenient and cost-effective for the patient and health careprofessionals.

[0183] Where combined administration of a chemotherapeutic agent (otherthan an antracycline) is desired, the combined administration includescoadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the antibody or may be given simultaneouslytherewith. The antibody may be combined with an anti-estrogen compoundsuch as tamoxifen or an anti-progesterone such as onapristone (see, EP616 812) in dosages known for such molecules.

[0184] It maybe desirable to also administer antibodies against othertumor associated antigens, such as antibodies which bind to the EGFR,ErbB3, ErbB4, or vascular endothelial growth factor (VEGF).Alternatively, or additionally, two or more anti-ErbB2 antibodies may beco-administered to the patient. Sometimes, it may be beneficial to alsoadminister one or more cytokines to the patient. The ErbB2 antibody maybe co-administered with a growth inhibitory agent. For example, thegrowth inhibitory agent may be administered first, followed by the ErbB2antibody. However, simultaneous administration, or administration of theErbB2 antibody first is also contemplated. Suitable dosages for thegrowth inhibitory agent are those presently used and may be lowered dueto the combined action (synergy) of the growth inhibitory agent andanti-ErbB2 antibody.

[0185] In addition to the above therapeutic regimens, the patient may besubjected to surgical removal of cancer cells and/or radiation therapy.

[0186] For the prevention or treatment of disease, the appropriatedosage of anti-ErbB2 antibody will depend on the type of disease to betreated, as defined above, the severity and course of the disease,whether the antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments. Where the treatment involves a series oftreatments, the initial dose or initial doses are followed at daily orweekly intervals by maintenance doses. Each maintenance dose providesthe same or a smaller amount of antibody compared to the amount ofantibody administered in the initial dose or doses.

[0187] Depending on the type and severity of the disease, about 1 μg/kgto 15 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

[0188] According to the invention, dosage regimens may include aninitial dose of anti-ErbB2 of 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered byintravenous or subcutaneous infusion, followed by subsequent weeklymaintenance doses of 2 mg/kg by intravenous infusion, intravenous bolusinjection, subcutaneous infusion, or subcutaneous bolus injection. Wherethe antibody is well-tolerated by the patient, the time of infusion maybe reduced.

[0189] Alternatively, the invention includes an initial dose of 12 mg/kganti-ErbB2 antibody, followed by subsequent maintenance doses of 6 mg/kgonce per 3 weeks.

[0190] Another dosage regimen involves an initial dose of 8 mg/kganti-ErbB2 antibody, followed by 6 mg/kg once per 3 weeks.

[0191] Still another dosage regimen involves an initial dose of 8 mg/kganti-ErbB2 antibody, followed by subsequent maintenance doses of 8 mg/kgonce per week or 8 mg/kg once every 2 to 3 weeks.

[0192] As an alternative regimen, initial doses of 4 mg/kg anti-ErbB2antibody may be administered on each of days 1, 2 and 3, followed bysubsequent maintenance doses of 6 mg/kg once per 3 weeks.

[0193] An additional regimen involves an initial dose of 4 mg/kganti-ErbB2 antibody, followed by subsequent maintenance doses of 2 mg/kgtwice per week, wherein the maintenance doses are separated by 3 days.

[0194] Alternatively, the invention may include a cycle of dosing inwhich delivery of anti-ErbB2 antibody is 2-3 times per week for 3 weeks.The 3 week cycle is preferably repeated as necessary to achievesuppression of disease symptoms.

[0195] The invention further includes a cyclic dosage regimen in whichdelivery of anti-ErbB2 antibody is daily for 5 days. According to theinvention, the cycle is preferably repeated as necessary to achievesuppression of disease symptoms. Further information about suitabledosages is provided in the Examples below.

[0196] VI. Articles of Manufacture

[0197] In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container, alabel and a package insert. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is effective for treating the condition and may have asterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is ananti-ErbB2 antibody. The label on, or associated with, the containerindicates that the composition is used for treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes. In addition, the article of manufacture may comprise a packageinserts with instructions for use, including, e.g., a warning that thecomposition is not to be used in combination with anthacycline-typechemotherapeutic agent, e.g. doxorubicin or epirubicin.

[0198] Deposit of Materials

[0199] The following hybridoma cell lines have been deposited with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.,USA (ATCC): Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215Oct. 17, 1996 7F3 ATCC HB-12216 Oct. 17, 1996 4D5 ATCC CRT 10463 May 24,1990 2C4 ATCC HB-12697 Apr. 8, 1999

[0200] Further details of the invention are illustrated by the followingnon-limiting Examples. cl EXAMPLES

Example 1

[0201] Preparation and Efficacy of HERCEPTIN® Anti-ErbB2 AntibodyMaterials and Methods

[0202] Anti-ErbB2 monoclonal antibody The anti-ErbB2 IgG₁κ murinemonoclonal antibody 4D5, specific for the extracellular domain of ErbB2,was produced as described in Fendly et al., Cancer Research 5o:1550-1558 (1990) and WO89/06692. Briefly, NIH 3T3/HER2-3₄₀₀ cells(expressing approximately 1×10⁵ ErbB2 molecules/cell) produced asdescribed in Hudziak et al., Proc. Natl. Acad. Sci. (USA) 84:7159 (1987)were harvested with phosphate buffered saline (PBS) containing 25 mMEDTA and used to immunize BALB/c mice. The mice were given injectionsi.p. of 10⁷ cells in 0.5 ml PBS on weeks, 0, 2, 5 and 7. The mice withantisera that immunoprecipitated ³²P-labeled ErbB2 were given i.p.injections of a wheat germ agglutinin-Sepharose (WGA) purified ErbB2membrane extract on weeks 9 and 13. This was followed by an i.v.injection of 0. 1 ml of the ErbB2 preparation and the splenocytes werefused with mouse myeloma line X63-Ag8.653. Hybridoma supernatants werescreened for ErbB2-binding by ELISA and radioimmunoprecipitation.MOPC-21 (IgG1), (Cappell, Durham, N.C.), was used as an isotype-matchedcontrol.

[0203] The treatment was performed with a humanized version of themurine 4D5 antibody (HERCEPTIN® anti-ErbB2 antibody). The humanizedantibody was engineered by inserting the complementarity determiningregions of the murine 4D5 antibody into the framework of a consensushuman immunoglobulin IgG₁ (IgG₁) (Carter et al., Proc. Natl. Acad. Sci.USA 89:4285-4289 [1992]). The resulting humanized anti-ErbB2 monoclonalantibody has high affinity for p185^(HER2) (Dillohiation constant[K_(d)]=0.1 nmol/L), markedly inhibits, in vitro and in humanxenografts, the growth of breast cancer cells that contain high levelsof p185^(HER2), induces antibody-dependent cellular cytotoxicity (ADCC),and has been found clinically active, as a single agent, in patientswith ErbB2-overexpressing metastatic breast cancers that had receivedextensive prior therapy. HERCEPTIN® anti-ErbB2 antibody is produced by agenetically engineered Chinese Hamster Ovary (CHO) cell line, grown inlarge scale, that secretes the antibody into the culture medium. Theantibody is purified from the CHO culture media using standardchromatographic and filtration methods. Each lot of antibody used inthis study was assayed to verify identity, purity, and potency, as wellas to meet Food and Drug Administration requirements for sterility andsafety.

[0204] Eligibility Criteria Patients had to fulfill all of the followingcriteria to be eligible for study admission:

[0205] Metastatic breast cancer

[0206] Overexpression of the ErbB2 (HER2) oncogene (2+ to 3+ asdetermined by immunohistochemistry or fluorescence in situ hybridization(FISH). [Tumor expression of ErbB2 can be determined byimmunohistochemical analysis, as previously described (Slamon et al.,[1987] and [1989], supra), of a set of thin sections prepared from thepatient's paraffin-archived tumor blocks. The primary detecting antibodyused is murine 4D5 MAb, which has the same CDRs as the humanizedantibody used for the treatment. Tumors are considered to overexpressErbB2 if at least 25% of tumor cells exhibit characteristic membranestaining for p185^(HER2)].

[0207] Bidimensionally measurable disease (including lytic bone lesions)by radiographic means, physical examination, or photographs

[0208] Measurable disease was defined as any mass reproduciblymeasurable in two perpendicular diameters by physical examination, X-ray(plain films), computerized tomography (CT), magnetic resonance imaging(MRI), ultrasound, or photographs.

[0209] Osteoblastic metastases, pleural effusions, or ascites were notconsidered to be measurable. Measurable lesions must be at least 1 cm ingreatest dimension. Enumeration of evaluable sites of metastatic diseaseand number of lesions in an evaluable site (e.g. lung) had to berecorded on the appropriate Case Report Form (CRF). If a large number ofpulmonary or hepatic lesions were present, the six largest lesions persite were followed.

[0210] The ability to understand and willingness to sign a writteninformed consent form

[0211] Women>18 years

[0212] Suitable candidates for receiving concomitant cytotoxicchemotherapy as evidenced by screening laboratory assessments ofhematologic, renal, hepatic, and metabolic functions.

[0213] Exclusion Criteria Patients with any of the following wereexcluded from study entry:

[0214] Prior cytotoxic chemotherapy for metastatic breast cancer

[0215] Patients may have received prior hormonal therapy (e.g.tamoxifen) for metastatic disease or cytotoxic therapy in the adjuvantsetting.

[0216] Concomitant malignancy that has not been curatively treated

[0217] A performance status of <60% on the Kamofsky scale

[0218] Pregnant or nursing women; women of childbearing potential,unless using effective contraception as determined by the investigator

[0219] Bilateral breast cancer (either both primary tumors must have 2+to 3+HER2 overexpression, or the metastatic site must have 2+ to 3+HER2overexpression)

[0220] Use of investigational or unlicensed agents within 30 days priorto study entry

[0221] Clinically unstable or untreated metastases to the brain (e.g.requiring radiation therapy)

[0222] Based upon the foregoing criteria, 469 patients were chosen, andenrolled in the study. Half the patients (stratified by chemotherapy)were randomized to additionally receive the HERCEPTIN® anti-ErbB2antibody (see below).

[0223] Administration and Dosage

[0224] Anti-ErbB2 Antibody

[0225] On day 0, a 4 mg/kg dose of humanized anti-ErbB2 antibody(HERCEPTIN®, H) was administered intravenously, over a 90-minute period.Beginning on day 7, patients received weekly administration of 2 mg/kgantibody (i.v.) over a 90-minute period.

[0226] Chemotherapy

[0227] The patients received one of two chemotherapy regimens for aminimum of six cycles, provided their disease was not progressing: a)cyclophosphamide and doxorubicin or epirubicin (AC), if patients havenot received anthracycline therapy in the adjuvant setting, or b)paclitaxel (T, TAXOL®), if patients have received any anthracyclinetherapy in the adjuvant setting. The initial dose of the HERCEPTIN®anti-ErbB2 antibody preceded the first cycle of either chemotherapyregimen by 24 hours. Subsequent doses of the antibody were givenimmediately before chemotherapy administration, if the initial dose ofthe antibody was well tolerated. If the first dose of the antibody wasnot well tolerated, subsequent infusions continued to precedechemotherapy administration by 24 hours. Patients were permitted tocontinue receiving chemotherapy beyond six cycles if, in the opinion ofthe treating physician, they were continuing to receive treatmentbenefit.

[0228] Cyclophosphamide (600 mg/m²) was given either by iv push over aminimum period of 3 minutes or by infusion over a maximum period of 2hours.

[0229] Doxorubicin (60 mg/m²) or epirubicin (75 mg/m²) were given eitherby slow iv push over a minimum period of 3-5 minutes or by infusion overa maximum period of 2 hours, according to institutional protocol.

[0230] Paciltaxel (TAXOL®) was given at a dose of 175 mg/m² over 3 hoursby intravenous administration. All patients receiving paclitaxel werepremedicated with dexamethasone (or its equivalent) 20 mg×2,administered orally 12 and 6 hours prior to paclitaxel; diphenhydramine(or its equivalent) 50 mg, iv, administered 30 minutes prior topaclitaxel, and dimetidine (or another H₂ blocker) 300 mg, iv,administered 30 minutes prior to paclitaxel.

[0231] Response Criteria

[0232] Progressive Disease Objective evidence of an increase of 25% ormore in any measurable lesion. Progressive disease also includes thoseinstances when new lesions have appeared. For bone lesions, progressionis defined as a 25% increase in objective measurement by plain film, CT,MRI; symptomatic new lesions not due to fracture; or requirement forpalliative radiotherapy.

[0233] Complete Response Disappearance of all radiographically and/orvisually apparent tumor for a minimum of 4 weeks. Skin and chest wallcomplete responses had to be confirmed by biopsy.

[0234] Partial Response A reduction of at least 50% in the sum of theproducts of the perpendicular diameters of all measurable lesions for aminimum period of 4 weeks. No new lesions may have appeared, nor may anylesions have progressed in size.

[0235] Minor Response A reduction of 25% to 49% in the sum of theproducts of the perpendicular diameters of all measurable lesions. Nonew lesions may have appeared, nor may any lesions have progressed insize.

[0236] Stable Disease No change of greater than 25% in the size ofmeasurable lesions. No lesions may have appeared.

[0237] Time to disease progression (TTP) was calculated from thebeginning of therapy to progression. Confidence limits for responserates were calculated using the exact method for a single proportion.(Fleiss, J L, Statistical Methods for Rates and Proportions (ed.2), NewYork, N.Y., Wiley, 1981, pp 13-17).

[0238] Results

[0239] At a median follow-up of 10.5 months, assessments of time todisease progression (TTP in months) and response rates (RR) showed asignificant augmentation of the chemotherapeutic effect by HERCEPTIN®anti-ErbB2 antibody, without increase in overall severe adverse events(AE): TABLE 1 HERCEPTIN ® Anti-ErbB2 Antibody Efficacy EnrolledTTP(months) RR(%) AE(%) CRx 234 5.5 36.2 66 CRx + H 235 8.6* 62.00** 69AC 145 6.5 42.1 71 AC + H 146 9.0 64.9 68 T 89 4.2 25.0 59 T + H 89 7.157.3 70

[0240] A syndrome of myocardial dysfunction similar to that observedwith anthracyclines was reported more commonly with a combined treatmentof AC+H (18% Grade ¾) than with AC alone (3%), T (0%), or T+H (2%).

[0241] These data indicate that the combination of anti-ErbB2 antibodytreatment with chemotherapy markedly increases the clinical benefit, asassessed by response rates and the evaluation of disease progression.However, due to the increased cardiac side-effects of doxorubicin orepirubicin, the combined use of anthracyclines with anti-ErbB2 antibodytherapy is contraindicated. The results, taking into account risk andbenefit, favor treatment with HERCEPTIN® anti-ErbB2 antibody andpaclitaxel (TAXOL®) where a combined treatment regimen is desired.

Example 2

[0242] Pharmacokinetic and Pharmacodynamic Properties of Anti-ErbB2Antibody (HERCEPTIN®)

[0243] HERCEPTIN® anti-ErbB2 antibody was administered by intravenousinfusion to human patients selected according to the criteria providedin Example 1. An initial dose of 4 mg/kg HERCEPTIN® anti-ErbB2 antibodywas delivered by intravenous infusion, followed by subsequent i.v.infusions of 2 mg/kg HERCEPTIN® anti-ErbB2 antibody weekly for severalweeks. Two hundred thirteen patients began this treatment regimen andserum drug concentration was obtained beyond 8 weeks for fewer than 90patients as selective discontinuation of patients with rapidlyprogressing disease occurred. Of the 213 patients who began treatment,serum trough concentration data were available for 80 patients at Week12, for 77 patients at Week 16, for 44 patients at Week 20, for 51patients at Week 24, for 25 patients at Week 28, for 23 patients at Week32, and for 37 patients at Week 36.

[0244] HERCEPTIN® Anti-ErbB2 Antibody Trough Serum Concentrations forWeeks 0-36

[0245] The HERCEPTIN® anti-ErbB2 antibody trough serum concentrations(μg/ml, mean±SE) from Week 2 through Week 36 are plotted in FIG. 3 (darkcircles). The number of patients was fairly constant because data frompatients discontinued from the program due to rapidly progressingdisease were excluded from this analysis. Trough serum concentrationstended to increase through Week 12 and tended to plateau after thattime.

[0246] HERCEPTIN® Anti-ErbB2 Antibody Trough and Peak SerumConcentrations for Weeks 1-8

[0247] Some HERCEPTIN® anti-ErbB2 antibody serum concentration data wereavailable for 212 of the original 213 patients. Trough and peak serumconcentration data reflecting the first HERCEPTIN® anti-ErbB2 antibodyinfusion were available for 195 of the 212 patients. For the seventhinfusion, trough serum concentration data were available for 137/212patients and peak serum concentration data were available for 114/212patients. Table 2 presents a summary of statistics from trough and peakserum concentrations for the first 8 weeks of treatment. Peak sampleswere drawn shortly after the end of HERCEPTIN® anti-ErbB2 antibodyadministration; trough samples were drawn prior to the subsequent dose(i.e., 1 week later). Serum concentrations of HERCEPTIN® anti-ErbB2antibody were determined as disclosed herein. TABLE 2 HERCEPTIN ®Anti-ErbB2 Antibody Trough and Peak Serum Concentrations for the First 8Weeks of Treatment (μg/ml) Dose Number n Mean SD Minimun Maximum Peak 1195 100.3 35.2 30.7 274.6 Trough 195 25.0 12.7 0.16 60.7 Peak 2 190 74.331.3 20.8 307.9 Trough 167 30.4 16.0 0.2 74.4 Peak 3 167 75.3 26.8 16.1194.8 Trough 179 33.7 17.9 0.2 98.2 Peak 4 175 80.2 26.9 22.2 167 Trough132 38.6 20.1 0.2 89.4 Peak 5 128 85.9 29.2 27.8 185.8 Trough 141 42.124.8 0.2 148.7 Peak 6 137 87.2 32.2 28.9 218.1 Trough 115 43.2 24.0 0.2109.9 Peak 7 114 89.7 32.5 16.3 187.8 Trough 137 48.8 24.9 0.2 105.2Peak 8 133 95.6 35.9 11.4 295.6

[0248] The data in Table 2 suggest that there was an increase in troughserum concentration over time. Of the many patients studied, there were18 patients for whom the trough concentrations did not exceed 20 μg/mlfrom Week 2 through Week 8. A HERCEPTIN® anti-ErbB2 antibody troughserum concentration of 20 μg/ml was nominally targeted for these studiesbased on prior pharmacologic studies in animals and exploratory analysesin clinical trials.

[0249] Patient response status was evaluated relative to serumconcentration of HERCEPTIN® anti-ErbB2 antibody. For this purpose, meanserum concentration (an average of troughs and peaks) was calculated forvarious times and patient response status (where the patient responsestatus was determined by an independent Response Evaluation Committee).The increase in serum concentration between Weeks 2 and 8 appeared to begreater in responders than in nonresponders, suggesting that there is arelationship between response status and HERCEPTIN® anti-ErbB2 antibodyserum concentration. A statistical analysis (analysis of variance) oftrough serum concentration values at Week 2 and an average of Weeks 7and 8 in relation to response status indicated a highly significantrelationship between response status and average trough of Weeks 7 and 8(p<0.001). The results indicated that there was a significant differencebetween the trough serum concentration (average troughs of Weeks 7 and8) in the responders and nonresponders: trough concentrations were 60±20μg/ml in the responders versus 44±25 μg/ml in the nonresponders(mean±SD). HER2 overexpression level and type of metastatic sites wereassociated with significant differences in trough serum concentrations.At Week 2, patients with 2+ HER2 overexpression had significantly highertrough serum concentrations (n=40, mean=28.8 μg/ml, SD=10.4) comparedwith patients with 3+HER2 overexpression (n=155, mean=24.1 μg/ml,SD=13.1). This difference in the average trough serum concentrations forWeeks 7 and 8 was no longer statistically significant. Further, at Week2, patients with superficial disease had significantly higher troughserum concentrations (n=12, mean 34.1 μg/ml, SD=12.0) compared withpatients with visceral disease (n=183, mean=24.4 μg/ml, SD=12.6). Thisdifference in the average trough serum concentrations for Weeks 7 and 8was significant. These data indicate that the rise in trough serumconcentrations between Weeks 2 and 7/8 occurs for human patients withvarious disease profiles.

[0250] In a subsequent, similarly designed study, human breast cancerpatients were treated with a loading dose of 8 mg/kg followed bymaintenance doses of4 mg/kg weekly. The results of this preliminaryhuman study indicated that an 8 mg/kg load:4 mg/kg weekly maintenanceregimen was efficacious in reducing tumor volume in the patients.

[0251] The data disclosed in this Example indicate that front loading ofantibody, such that a target serum concentration is reached morequickly, may be associated with improved outcomes.

Example 3

[0252] I.V. Bolus Delivery and Subcutaneous Infusion of HERCEPTIN®Anti-ErbB2 Antibody Effectively Decrease Tumor Volume in the Mouse.

[0253] The efficacy of infusion or bolus delivery of humanizedanti-ErbB2 antibody (HERCEPTIN®, see Example 1 for preparation), eitherby intravenous injection or subcutaneous injection, was examined. Thepurpose of the study was to ask whether subcutaneous delivery wasfeasible and whether the convenient subcutaneous bolus delivery wasuseful in treating metastatic breast cancer in animals inoculated with acell line that overexpresses the HER2 gene. The results, detailed below,show that i.v. and s.c. infusion and bolus delivery are feasibletreatment methodologies.

[0254] A study in a nude mouse xenograft model, which incorporates ahuman breast cancer cell line that naturally overexpresses the HER2 gene(BT-474M1, derived from BT-474 cells, ATCC Accession number HTB-20),comparing tumor volume as a function of i.v. bolus versus s.c. infusionwas performed as follows. In the first study athymic nude nu nu 7-9 weekold female mice were obtained from Taconic Inc (Germantown, N.Y.). Toinitiate tumor development, each mouse was inoculated subcutaneouslywith 3×10⁶ BT474M1 cells suspended in Matrigel™. When tumor nodulesreached a volume of approximately 100 mm³, animals were randomized to 4treatment groups. The groups were treated according to Table 3. TABLE 3Animal Groups and Doses for Comparison of I.V. Bolus and S.C. InfusionGroup, Target Loading Dose, Serum Conc. Route of Dose MaintenanceAntibody μg/ml Administration (mg/kg) Dose 1 - Control, 20 IV LD and2.20 0.250 mg/ml rhuMAb E25 SC infusion (infusate) 2 - Low Dose SC  1 IVLD and 0.313 0.050 mg/ml rhuMAb HER2 SC infusion (infusate) 3 - HighDose SC 20 IV LD and 6.25  1.00 mg/ml rhuMAb HER2 SC infusion (infusate)4 - IV Multi-Dose 20 IV LD and MD 4.00    2 mg/kg/ rhuMAb HER2 (trough)week (IV bolus)

[0255] Animals were exposed to estrogen by subcutaneous sustainedrelease estrogen pellet 9 days before the start of dosing to promotegrowth of grafted tumor cells. The animals were inoculated with theBT474M 1 cells 8 days before the beginning of treatment and tumors wereallowed to grow. The animals were then treated with nonrelevant antibodyE25 (non-specific for HER2 receptor, but a member of the monoclonal IgGclass) or test antibody HERCEPTIN® anti-ErbB2 anitbody as indicated inTable 3. The dosage levels were selected to achieve target serumconcentrations of HERCEPTIN®, either 1 μg/ml or 20 μg/ml, bysubcutaneous pump infusion or by i.v. bolus delivery. The study groupswere treated until day 35. The serum concentration of HERCEPTIN®anti-ErbB2 antibody was measured weekly (just prior to dosing for Group4) using 3 mice/group/time point. The anti-ErbB2 antibody concentrationwas determined according to the method disclosed herein involvingstandard techniques. Tumor volumes were measured two days before dosingbegan and twice per week from day 6 to day 35 in the study for whichdata is tabulated below. Tumors were measured in three dimensions andvolumes were expressed in mm³. Efficacy was determined by a statisticalcomparison (ANOVA) of tumor volumes of test animals relative tountreated control animals.

[0256] As shown in Table 4, below, treatment of the BT474M 1tumor-bearing mice with HERCEPTIN® anti-ErbB2 antibody by the indicateddosage methods significantly inhibited the growth of the tumors. AllHERCEPTIN®-treated groups showed similar inhibition of tumor growthrelative to the control group. No dose-response was observed. TABLE 4Comparison of S.C. Infusion and I.V. Bolus Delivery Tumor VolumeHERCEPTIN ® Tumor Volume (area under curve) Serum Conc. (mm³), Day 35,Day 6-Day 35 (μg/ml), Day 27, Treatment Group (n = 14) (n = 13) (n = 3)control s.c. 764 ± 700 5650 ± 4700 4.16 ± 1.94 infusion s.c. infusion80.6 ± 158  1610 ± 1250 2.11 ± 1.74 (low dose) s.c. infusion   31 ± 75.61440 ± 1140 22.1 ± 5.43 (high dose) i.v. bolus dose* 49.7 ± 95.7 2150 ±1480 21.7 ± 17.1**

[0257] The results tabulated above indicate that maintenance of a serumconcentration of approximately 2 μg/ml was as effective as aconcentration of 20 μg/ml in this study. The results indicated thatdosing by subcutaneous infusion was as effective as intravenous bolusdosing and achieved similar trough serum concentrations. The resultsalso indicate that the dose levels studied are at the top of thedose-response curve in this model and that subcutaneous dosing iseffective in treating breast cancer tumors. Thus, subcutaneousadministration of maintenance doses is feasible as part of a HERCEPTIN®anti-ErbB2 antibody treatment regimen.

Example 4

[0258] I.V. Bolus and Subcutaneous Bolus Deliveries of HERCEPTIN®Anti-ErbB2 Antibody Effectively Decrease Tumor Volume in the Mouse.

[0259] Subcutaneous bolus delivery is convenient and cost-effective forthe patient and health care professionals. The results of the studydisclosed in this example indicate that subcutaneous bolus delivery wasas effective as intravenous bolus delivery in reducing breast cell tumorsize in a mouse.

[0260] This study was set up as disclosed herein in Example 3 for thecomparison of intravenous bolus and subcutaneous infusion delivery. Asustained release estrogen implant was inserted subcutaneously one daybefore tumor cell innoculation as described in Example 3. Six days aftertumor cell innoculation, the initial tumor measurement was performed.Seven days after tumor cell innoculation, the first dose of controlantibody or HERCEPTIN® anti-ErbB2 antibody was delivered. The animalgroups, type of delivery, loading dose and maintenance doses areprovided in Table 4. Animals were dosed once weekly for 4 weeks. TABLE 5Animal Groups and Doses for Comparison of I.V. Bolus and S.C. BolusDelivery Maintenance Route of Ad- Loading Dose Dose Group ministration(mg/kg) (mg/kg/week) n 1 - Control IV 8 4 10 rhuMAb E25 2 - rhuMAb HER2IV 2 1 10 3 - rhuMAb HER2 IV 4 2 10 4 - rhuMAb HER2 IV 8 4 10 5 - rhuMAbHER2 SC 4 2 10

[0261] The mice were treated according to the information in Table 4 andusing the techniques disclosed in Example 3. The serum concentration ofHERCEPTIN® anti-ErbB2 antibody was measured weekly before each weeklyi.v. maintenance dose according to the procedure described herein andusing standard techniques. The control E25 antibody serum concentrationwas determined according to standard immunoassay techniques. Table 6shows the increase in HERCEPTIN® anti-ErbB2 antibody serumconcentrations with time. TABLE 6 IV versus SC Bolus Delivery: SerumHERCEPTIN ® Anti-ErbB2 Antibody Concentration Serum Concentration, μg/mlDay 0 Day 7 Day 14 Day 21 Treatment Group Mean Mean Mean Mean (delivery,MD) (SD) (SD) (SD) (SD) 1 - Control rhu MAb E25 0 25.9 34.6 38.5 (IV, 4mg/kg) (0) (8.29) (11.2) (14.4) 2 - rhu MAb HER2 0 4.96 8.55 8.05 (IV, 1mg/kg) (0) (3.79) (5.83) (4.67) 3 - rhu MAb HER2 0 13.4 18.9 22.6 (IV, 2mg/kg) (0) (9.24) (12.0) (9.21) 4 - rhu MAb HER2 0 29.6 37.7 46.2 (IV, 4mg/kg) (0) (13.5) (14.4) (13.8) 5 - rhu MAb HER2 0 12.5 16.9 17.6 (SC, 2mg/kg) (0) (7.33) (10.2) (10.7)

[0262] Table 7 shows the relative efficacy of intravenous bolus deliveryand subcutaneous bolus delivery for Groups 1-5 having achieved the serumantibody concentrations presented in Table 6. For this study, efficacywas measured as a decrease in tumor volume. Tumor volume was measuredtwice weekly. TABLE 7 Efficacy of HERCEPTIN ® Anti-ErbB2 AntibodyMeasured as a Change in Tumor Volume Comparing Intravenous Bolus andSubcutaneous Bolus Delivery, Mean (SD) Treatment Tumor Tumor Vol. Day6-Day 31* Tumor Growth Group Tumor Vol. Vol. Day Day 31, Area UnderCurve Rate (Delivery, MD) Day 6, mm³ 28, mm³ mm³ Tumor Vol., mm³ on Log(TM + 1) 1-IV Control 321 1530 1630 13600 0.0660 (190) (1040) (1170)(7230) (0.0200) 2-IV Herceptin 297 175 151 4690 −0.0505 1 mg/kg (130)(215) (188) (1400) (0.142) 3-IV Herceptin 269 75.7 73.6 3510 −0.0608 2mg/kg (129) (92.4) (84.5) (1220) (0.110) 4-IV Herceptin 272 25.3 25.82880 −0.0810 4 mg/kg (117) (75.9) (72.9) (1230) (0.0859) 5-SC Herceptin268 76.2 90.4 3230 −0.0304 2 mg/kg (117) (98.8) (105) (1440) (0.104)

[0263]FIGS. 4A and 4B are graphical plots of changes in tumor volumeover time, some of which data is found in Table 7. FIG. 4A is a linearplot of tumor volume versus time. FIG. 4B is a semilogarithmic plot ofthe same data, allowing the test points be viewed more clearly. The datain Table 7 and FIGS. 4A and 4B indicate that, although a dose-relatedresponse was not observed between HERCEPTIN-treated groups, dosing bysubcutaneous bolus was as effective as intravenous bolus dosing andachieved similar trough serum concentrations.

Example 5

[0264] Regimens for Intravenous and Subcutaneous Delivery of Anti-ErbB2Antibody.

[0265] According to the invention, methods of anti-ErbB2 antibody (e.g.,HERCEPTIN®) delivery comprise greater front loading of the drug toachieve a target serum concentration in approximately 4 weeks or less,preferably 3 weeks or less, more preferably 2 weeks or less, and mostpreferably 1 week or less, including one day or less. According to theinvention, this initial dosing is followed by dosing that maintains thetarget serum concentration by subsequent doses of equal or smalleramount. An advantage of the methods of the invention is that themaintenance dosing may be less frequent and/or delivered by subcutaneousinjection, making the treatment regimens of the invention convenient andcost-effective for the patient and medical professionals administeringthe antibody. In addition, a subcutaneous maintenance dose regimen maybe interrupted by intravenous dosing (such as infusion) when thepatient's chemotherapy requires delivery of other drugs by intravenousinjection.

[0266] To test the following dosage regimens, human subjects areselected according to the criteria disclosed in Example 1, above. Thenumber of initial doses is one or more doses sufficient to achieve anefficacious target serum concentration in approximately 4 weeks or less,preferably 3 weeks or less, more preferably 2 weeks or less, and mostpreferably 1 week or less, including 1 day or less. The number ofmaintenance doses may be one or more doses sufficient to achievesuppression of disease symptoms, such as a decrease in tumor volume. Themaintenance doses are equal to or smaller than the initial dose ordoses, consistent with an object of the invention of administeringHERCEPTIN® anti-ErbB2 antibody by regimens providing greater frontloading. The specific drug delivery regimens disclosed herein arerepresentative of the invention and are not meant to be limiting.

[0267] In one trial, an initial dose of 6 mg/kg, 8 mg/kg, or 12 mg/kg ofHERCEPTIN® anti-ErbB2 antibody is delivered to human patients byintravenous or subcutaneous injection. Initial doses (loading doses) aredelivered by intravenous infusion or bolus injection or preferablysubcutaneous bolus injection. Preferably a target trough serumconcentration of HERCEPTIN® anti-ErbB2 antibody of approximately 10-20μg/ml is achieved (averaged for all patients in the treatment group) andmaintained by subsequent doses of anti-ErbB2 antibody that are equal toor smaller than the initial dose. In one method, a target trough serumconcentration is achieved and maintained by once-per-week deliveries of2 mg/kg HERCEPTIN® anti-ErbB2 antibody by intravenous or subcutaneousinjection for at least eight weeks. Alternatively, for this or anydosage regimen disclosed herein, subcutaneous continuous infusion bysubcutaneous pump is used to delivery subsequent maintenance doses.

[0268] In another method, an initial (front loading) dose of 8 mg/kgHERCEPTIN® anti-ErbB2 antibody is delivered by intravenous injection(infusion or bolus injection) or by subcutaneous bolus injection. Thisis followed by intravenous bolus injections, intravenous infusion,subcutaneous infusion, or subcutaneous bolus injection of 6 mg/kg at3-week intervals to maintain a trough serum concentration ofapproximately 10-20 μg/ml, averaged for an entire treatment group.

[0269] In another method, an initial (front loading) dose of 12 mg/kgHERCEPTIN® anti-ErbB2 antibody is delivered by intravenous injection(infusion or bolus injection) or by subcutaneous bolus injection. Thisis followed by intravenous bolus injections, intravenous infusion,subcutaneous infusion, or subcutaneous bolus injection of 6 mg/kg at3-week intervals to maintain a trough serum concentration ofapproximately 10-20 μg/ml.

[0270] In yet another method, an initial (front loading) dose of 8 mg/kgHERCEPTIN® anti-ErbB2 antibody is delivered by intravenous infusion orbolus injection, or preferably by subcutaneous bolus injection orinfusion. This is followed by administration of 8 mg/kg per week or 8mg/kg per 2-3 weeks to maintain a trough serum concentration ofHERCEPTIN® anti-ErbB2 antibody of approximately 10-20 μg/ml. Maintenancedoses are delivered by intravenous infusion or bolus injection, orpreferably by subcutaneous infusion or bolus injection.

[0271] In another method, the front loading initial dose is a series ofintravenous or subcutaneous injections, for example, one on each of days1, 2, and 3 of at least 1 mg/kg for each injection (where the amount ofanti-ErbB2 antibody delivered by the sum of initial injections is morethan 4 mg/kg) , followed by maintenance doses of 6 mg/kg once each 3week interval to maintain a target trough serum concentration (forexample, approximately 10-20 μg/ml) of HERCEPTIN® anti-ErbB2 antibody.The maintenance doses are delivered by intravenous infusion or bolusinjection or by subcutaneous infusion or subcutaneous bolus injection.

[0272] In yet another method, the front loading is by intravenousinfusion of at least 1 mg/kg, preferably 4 mg/kg on each of fiveconsecutive days, followed by repeats of this cycle a sufficient numberof times to achieve suppression of disease symptoms. Following theinitial dose or doses, subsequent doses may be delivered by subcutaneousinfusion or bolus injection if tolerated by the patient. Suchsubcutaneous delivery is convenient and cost-effective for the patientand administering health care professionals.

[0273] In still another method, HERCEPTIN® anti-ErbB2 antibody isdelivered initially as at least 2 intravenous infusions per week forthree weeks, followed by repeats of this cycle to maintain anefficacious trough serum concentration of HERCEPTIN® anti-ErbB2antibody. The dose is at least 4 mg/kg of anti-ErbB2 antibody,preferably at least 5 mg/kg. The maintenance drug deliveries may beintravenous or subcutaneous.

[0274] Where the animal or patient tolerates the antibody during andafter an initial dose, delivery of subsequent doses may be subcutaneous,thereby providing greater convenience and cost-effectiveness for thepatient and health care professionals.

[0275] In animal studies, an initial dose of more than 4 mg/kg,preferably more than 5 mg/kg delivered by intravenous or subcutaneousinjection, is followed by subcutaneous bolus injections of 2 mg/kg twiceper week (separated by 3 days) to maintain a trough serum concentrationof approximately 10-20 μg/ml. In addition, where the animal or patientis known to tolerate the antibody, an initial dose of HERCEPTIN®anti-ErbB2 antibody is optionally and preferably deliverable bysubcutaneous bolus injection followed by subcutaneous maintenanceinjections.

[0276] While target serum concentrations are disclosed herein for thepurpose of comparing animal studies and human trials, target serumconcentrations in clinical uses may differ. The disclosure providedherein guides the user in selecting a front loading drug deliveryregimen that provides an efficacious target trough serum concentration.

[0277] The methods of the invention disclosed herein optionally includethe delivery of HERCEPTIN® anti-ErbB2 antibody in combination with achemotherapeutic agent (other than an anthrocycline derivative) toachieve suppression of disease symptoms. The chemotherapeutic agent maybe delivered with HERCEPTIN® anti-ErbB2 antibody or separately andaccording to a different dosing schedule. For example, subcutaneousdelivery of HERCEPTIN® anti-ErbB2 antibody with TAXOL® is included inthe invention. In addition, intravenous or subcutaneous injection of 8mg/kg HERCEPTIN® anti-ErbB2 antibody, followed by intravenous orsubcutaneous injection of 6 mg/kg HERCEPTIN® anti-ErbB2 antibody every 3weeks is administered in combination with a chemotherapeutic agent, suchas a taxoid (e.g. paclitaxel 175 mg/m2 every 3 weeks) or ananthracycline derivative (e.g. doxorubicin 60 mg/m2 or epirubicin 75mg/m2 every 3 weeks). Optionally, where an anthracycline derivative isadministered, a cardioprotectant (e.g. 600 mg/m2 cyclophosphamide every3 weeks) is also administered. In another combination therapy,anti-ErbB2 antibody is administered in a loading dose of more than 4mg/kg, preferably more than 5 mg/kg, and more preferably at least 8mg/kg. The loading dose is followed by maintenance doses of at least 2mg/kg weekly, preferably 6 mg/kg every 3 weeks. The combination therapyincludes administration of a taxoid during treatment with anti-ErbB2antibody. According to one embodiment of the invention, the taxoid ispaclitaxel and is administered at a dose of 70-100 mg/m²/week. Accordingto another embodiment of the invention, the taxoid is docetaxel and isadministered at a dose of 30-70 mg/m²/week.

Example 6

[0278] HERCEPTIN® Administered Intravenously Every Three Weeks inCombination with Paclitaxel

[0279] Currently, the recommended dose of HERCEPTIN® is 2 mg/kg onceweekly. Patients will be administered HERCEPTIN® every three weeksinstead of weekly, along with paclitaxel (175 mg/m² every three weeks).Simulation of the proposed treatment regimen suggests that the troughserum concentrations will be 17 mcg/ml, in the range (10-20 mcg/ml) ofthe targeted trough serum concentrations from previous HERCEPTIN® IVclinical trials. After the first 12 patients the PK parameters will beassessed, if exposure is felt inadequate, then the dose will beincreased to 8 mg/kg every three weeks for the remaining 12 patients.

[0280] Inclusion Criteria

[0281] 1) Females24 18 years of age

[0282] 2) Histologically confirmed ErbB2 over-expressing metastaticbreast cancer

[0283] 3) Patients who have been newly diagnosed with metastatic disease

[0284] 4) Have a Karnofsky performance status of 24 70%

[0285] 5) Give written informed consent prior to any study specificscreening procedures with the understanding that the patient has theright to withdraw from the study at any time, without prejudice.

[0286] Exclusion Criteria

[0287] 1) Pregnant or lactating women

[0288] 2) Women of childbearing potential unless (1) surgically sterileor (2) using adequate measures of contraception such as oralcontraceptive, intra-uterine device or barrier method of contraceptionin conjunction with spermicidal jelly.

[0289] 3) Clinical or radiologic evidence of CNS metastases.

[0290] 4) History of any significant cardiac disease

[0291] 5) LVEF≦50%

[0292] 6) No prior taxane therapy in any treatment setting.

[0293] 7) Any of the following abnormal baseline hematologic values:

[0294] Hb less than 9 g/dl

[0295] WBC less than 3.0×10⁹/l

[0296] Granulocytes less than 1.5×10⁹/l

[0297] Platelets less than 100×10⁹/l

[0298] 8) Any of the following abnormal baseline liver function tests:

[0299] Serum bilirubin greater than 1.5×ULN (upper normal limit)

[0300] ALT and/or AST greater than 2.5×ULN ( greater than 4.0×ULN ifliver or bone metastasis)

[0301] Alkaline phosphatase greater than 2.5×ULN ( greater than 4.0×ULNif liver or bone metastasis)

[0302] 9) The following abnormal baseline renal function tests:

[0303] serum creatinine greater than 1.5×ULN

[0304] 10) History of other serious medical conditions that wouldpreclude patient participation in an investigational study.

[0305] HERCEPTIN® Loading dose and schedule: 8 mg/kg for first dose.Maintenance dose and schedule: 6 mg/kg every 3 weeks.

[0306] Paclitaxel—175 mg/m² IV every 3 weeks×6 cycles as a 3-hourinfusion.

[0307] NOTE: On the first cycle of treatment, paclitaxel will be dosed 8hours prior to HERCEPTIN® to determine the PK of paclitaxel alone.HERCEPTIN® will be administered 8 hours post-paclitaxel for the 1^(st)cycle only. In subsequent treatment cycles, HERCEPTIN® will beadministered prior to paclitaxel.

[0308] The total duration of this study is 18 weeks. Study subjects willreceive up to 6 total HERCEPTIN® doses. After the last subject hasreceived the last cycle of paclitaxel, data collection for safety andpharmacokinetic analysis will stop, and the study will close to protocolspecified treatment. Study subjects may continue to receive theHERCEPTIN® ±paclitaxel at the discretion of the investigator.

[0309] It is believed that the above treatment regimen will be effectivein treating metastatic breast cancer, despite the infrequency with whichHERCEPTIN® is administered to the patient.

[0310] While the particular aspects and embodiments of the invention asherein shown and disclosed in detail is fully capable of obtaining theobjects and providing the advantages herein before stated, it is to beunderstood that it is merely illustrative of some of the presentlypreferred embodiments of the invention and that no limitations areintended to the details of methods and articles of manufacture shownother than as described in the appended claims. The disclosures of allcitations in the specification are expressly incorporated herein byreference.

1 15 1 166 PRT Homo sapiens 1 Cys Thr Gly Thr Asp Met Lys Leu Arg LeuPro Ala Ser Pro Glu 1 5 10 15 Thr His Leu Asp Met Leu Arg His Leu TyrGln Gly Cys Gln Val 20 25 30 Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu ProThr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln GlyTyr Val Leu 50 55 60 Ile Ala His Asn Gln Val Arg Gln Val Pro Leu Gln ArgLeu Arg 65 70 75 Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala LeuAla 80 85 90 Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr95 100 105 Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu110 115 120 Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln125 130 135 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys140 145 150 Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg155 160 165 Ala 2 32 PRT Homo sapiens 2 Ser Thr Gln Val Cys Thr Gly ThrAsp Met Lys Leu Arg Leu Pro 1 5 10 15 Ala Ser Pro Glu Thr His Leu AspMet Leu Arg His Leu Tyr Gln 20 25 30 Gly Cys 3 11 PRT Artificialsequence salvage receptor binding epitope 3 Pro Lys Asn Ser Ser Met IleSer Asn Thr Pro 1 5 10 4 7 PRT Artificial sequence salvage receptorbinding epitope 4 His Gln Ser Leu Gly Thr Gln 1 5 5 8 PRT Artificialsequence salvage receptor binding epitope 5 His Gln Asn Leu Ser Asp GlyLys 1 5 6 8 PRT Artificial sequence salvage receptor binding epitope 6His Gln Asn Ile Ser Asp Gly Lys 1 5 7 8 PRT Artificial sequence salvagereceptor binding epitope 7 Val Ile Ser Ser His Leu Gly Gln 1 5 8 59 PRTHomo sapiens 8 Val Glu Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu TyrVal 1 5 10 15 Asn Ala Arg His Cys Leu Pro Cys His Pro Glu Cys Gln ProGln 20 25 30 Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val35 40 45 Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg 50 55 965 PRT Homo sapiens 9 Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn GlySer Val Thr 1 5 10 15 Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala CysAla His Tyr 20 25 30 Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser GlyVal Lys 35 40 45 Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp GluGlu 50 55 60 Gly Ala Cys Gln Pro 65 10 107 PRT Mus Musculus 10 Asp ThrVal Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val 1 5 10 15 Gly AspArg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 20 25 30 Ile Gly ValAla Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys 35 40 45 Leu Leu Ile TyrSer Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp 50 55 60 Arg Phe Thr Gly SerGly Ser Gly Thr Asp Phe Thr Phe Thr Ile 65 70 75 Ser Ser Val Gln Ala GluAsp Leu Ala Val Tyr Tyr Cys Gln Gln 80 85 90 Tyr Tyr Ile Tyr Pro Tyr ThrPhe Gly Gly Gly Thr Lys Leu Glu 95 100 105 Ile Lys 11 119 PRT Musmusculus 11 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr 2025 30 Asp Tyr Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu 35 4045 Glu Trp Ile Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr 50 55 60Asn Gln Arg Phe Lys Gly Lys Ala Ser Leu Thr Val Asp Arg Ser 65 70 75 SerArg Ile Val Tyr Met Glu Leu Arg Ser Leu Thr Phe Glu Asp 80 85 90 Thr AlaVal Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95 100 105 Phe AspTyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 110 115 12 107 PRTArtificial sequence humanized VL sequence 12 Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile ThrCys Lys Ala Ser Gln Asp Val Ser 20 25 30 Ile Gly Val Ala Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr ArgTyr Thr Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr TyrTyr Cys Gln Gln 80 85 90 Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly ThrLys Val Glu 95 100 105 Ile Lys 13 119 PRT Artificial sequence humanizedVH sequence 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln ProGly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr PheThr 20 25 30 Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu35 40 45 Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr 5055 60 Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser 65 7075 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95 100 105Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 14 107PRT Artificial sequence VL consensus sequence 14 Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr IleThr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30 Asn Tyr Leu Ala Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ala Ala Ser SerLeu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala ThrTyr Tyr Cys Gln Gln 80 85 90 Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln GlyThr Lys Val Glu 95 100 105 Ile Lys 15 119 PRT Artificial sequence VHconsensus sequence 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu ValGln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly PheThr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly LysGly Leu 35 40 45 Glu Trp Val Ala Val Ile Ser Gly Asp Gly Gly Ser Thr TyrTyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 8085 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Val Gly Tyr Ser Leu 95 100105 Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115

1. A method for the treatment of a human patient susceptible to ordiagnosed with a disorder characterized by overexpression of ErbB2receptor, comprising administering an effective amount of an anti-ErbB2antibody to the human patient, the method comprising: administering tothe patient an initial dose of at least approximately 5 mg/kg of theanti-ErbB2 antibody; and administering to the patient a plurality ofsubsequent doses of the antibody in an amount that is approximately thesame or less than the initial dose.
 2. The method of claim 1, whereinthe initial dose is at least approximately 6 mg/kg.
 3. The method ofclaim 2, wherein the initial dose is at least approximately 8 mg/kg. 4.The method of claim 3, wherein the initial dose is at leastapproximately 12 mg/kg.
 5. The method of claim 1, wherein the subsequentdoses are separated from each other in time by at least one week.
 6. Themethod of claim 1, wherein the subsequent doses are separated in timefrom each other by at least two weeks.
 7. The method of claim 1, whereinthe subsequent doses are separated in time from each other by at leastthree weeks.
 8. The method of claim 1, wherein the initial dose isadministered by intravenous injection, and wherein at least onesubsequent dose is administered by subcutaneous injection.
 9. The methodof claim 1, wherein the initial dose is administered by intravenousinjection, wherein at least two subsequent doses are administered, andwherein each subsequent dose is administered by a method selected fromthe group consisting essentially of intravenous injection andsubcutaneous injection.
 10. The method of claim 1, wherein the initialdose and at least one subsequent dose are administered by subcutaneousinjection.
 11. The method of claim 1, wherein the initial dose isselected from the group consisting essentially of approximately 6 mg/kg,8 mg/kg, or 12 mg/kg, wherein the plurality of subsequent doses are atleast approximately 2 mg/kg, and wherein the subsequent doses areseparated in time from each other by at least one week.
 12. The methodof claim 11, wherein the plurality of subsequent doses are separated intime from each other by at least two weeks.
 13. The method of claim 12,wherein the plurality of subsequent doses are separated in time fromeach other by at least three weeks.
 14. The method of claim 13, whereinthe initial dose is approximately 8 mg/kg, and wherein at least onesubsequent dose is approximately 6 mg/kg.
 15. The method of claim 13,wherein the initial dose is approximately 12 mg/kg, and wherein at leastone subsequent dose is approximately 6 mg/kg.
 16. The method of claim11, wherein the initial dose is approximately 8 mg/kg, and wherein atleast one subsequent dose is approximately 8 mg/kg.
 17. The method ofclaim 11, wherein the initial dose is approximately 8 mg/kg, wherein atleast one subsequent dose is 8 mg/kg, and wherein administration of theinitial dose and subsequent doses are separated in time by at least 2weeks.
 18. The method of claim 17, wherein the initial dose andsubsequent doses are separated in time by at least 3 weeks.
 19. Themethod of claim 1, wherein the initial dose is a plurality of doses,wherein each of the plurality of initial doses is at least approximately1 mg/kg and is administered on at least 2 consecutive days, wherein theat least one subsequent dose is at least approximately 2 mg/kg, andwherein administration of the last initial dose and the first subsequentand additional subsequent doses are separated in time by at least oneweek.
 20. The method of claim 19, wherein the initial dose is aplurality of doses, wherein each of the plurality of initial doses is atleast approximately 1 mg/kg and is administered on at least 3consecutive days, wherein the at least one subsequent dose is at leastapproximately 6 mg/kg, and wherein administration of the last initialdose and the first subsequent and additional subsequent doses areseparated in time by at least 3 weeks.
 21. The method of claim 1,wherein the initial dose is a plurality of doses, wherein each of theplurality of initial doses is at least approximately 1 mg/kg and isadministered at least twice per week for 3 weeks as a first dosagecycle, wherein the dosage cycle is repeated, and wherein the doses ofeach cycle are separated in time by at least 1 day, and wherein thedosage cycles are separated in time by at least 1 week.
 22. The methodof claim 1, wherein said disorder is a benign or malignant tumor. 23.The method of claim 1, wherein said disorder is a cancer.
 24. The methodof claim 23, wherein said cancer is selected from the group consistingof breast cancer, leukemia, squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer.
 25. The method of claim 24,wherein said cancer is breast cancer.
 26. The method of claim 25,wherein said cancer is metastatic breast carcinoma.
 27. The method ofclaim 1 wherein said antibody binds to the extracellular domain of theErbB2 receptor.
 28. The method of claim 27, wherein said antibodybindsto epitope 4D5 within the ErbB2 extracellular domain sequence.
 29. Themethod of claim 28, wherein said antibody is a humanized 4D5 anti-ErbB2antibody.
 30. The method of claim 1 wherein the method further comprisesadministering an effective amount of a chemotherapeutic agent.
 31. Themethod of claim 30, wherein the chemotherapeutic agent is a taxoid. 32.The method of claim 31, wherein said taxoid is paclitaxel or docetaxel.33. The method of claim 30, wherein the effective amount of theanti-ErbB2 antibody and the effective amount of the chemotherapeuticagent as a combination is lower than the sum of the effective amounts ofsaid anti-ErbB2 antibody and said chemotherapeutic agent, whenadministered individually, as single agents.
 34. The method of claim 30,wherein the chemotherapeutic agent is an anthrcycline derivative. 35.The method of claim 34, wherein the anthracycline derivative isdoxorubicin or epirubicin.
 36. The method of claim 34, wherein themethod further comprises administration of a cardioprotectant.
 37. Themethod of claim 1, wherein efficacy is measured by determining the timeto disease progression or the response rate.
 38. An article ofmanufacture, comprising a container, a composition within the containercomprising an anti-ErbB2 antibody, and a package insert containinginstructions to administer an initial dose of anti-ErbB2 antibody of atleast 5 mg/kg, and at least one subsequent dose that is the same amountor less than the initial dose.
 39. The article of manufacture of claim38, wherein the instructions are for administration of an initial doseby intravenous injection and at least one subsequent dose bysubcutaneous injection.
 40. The article of manufacture of claim 38,wherein the initial dose is at least approximately 6 mg/kg.
 41. Thearticle of manufacture of claim 40, wherein the initial dose is at leastapproximately 8 mg/kg.
 42. The article of manufacture of claim 41,wherein the initial dose is at least approximately 12 mg/kg.
 43. Thearticle of manufacture of claim 38, wherein the subsequent doses areseparated from each other in time by at least one week.
 44. The articleof manufacture of claim 38, wherein the subsequent doses are separatedin time from each other by at least two weeks.
 45. The article ofmanufacture of claim 38, wherein the subsequent doses are separated intime from each other by at least three weeks.
 46. The article ofmanufacture of claim 38, wherein the initial dose and at least onesubsequent dose are administered by subcutaneous injection.
 47. Thearticle of manufacture of claim 38, wherein the initial dose is selectedfrom the group consisting essentially of approximately 6 mg/kg, 8 mg/kg,or 12 mg/kg, wherein the plurality of subsequent doses are at leastapproximately 2 mg/kg, and wherein the subsequent doses are separated intime from each other by at least one week.
 48. The article ofmanufacture of claim 47, wherein the plurality of subsequent doses areseparated in time from each other by at least two weeks.
 49. The articleof manufacture of claim 48, wherein the plurality of subsequent dosesare separated in time from each other by at least three weeks.
 50. Thearticle of manufacture of claim 49, wherein the initial dose isapproximately 8 mg/kg, and wherein at least one subsequent dose isapproximately 6 mg/kg.
 51. The article of manufacture of claim 49,wherein the initial dose is approximately 12 mg/kg, and wherein at leastone subsequent dose is approximately 6 mg/kg.
 52. The article ofmanufacture of claim 47, wherein the initial dose is approximately 8mg/kg, and wherein at least one subsequent dose is approximately 8mg/kg.
 53. The article of manufacture of claim 47, wherein the initialdose is approximately 8 mg/kg, wherein at least one subsequent dose is 8mg/kg, and wherein administration of the initial dose and subsequentdoses are separated in time by at least 2 weeks.
 54. The article ofmanufacture of claim 53, wherein the initial dose and subsequent dosesare separated in time by at least 3 weeks.
 55. The article ofmanufacture of claim 38, wherein the initial dose is a plurality ofdoses, wherein each of the plurality of initial doses is at leastapproximately 1 mg/kg and is administered on at least 2 consecutivedays, wherein at least one subsequent dose is at least approximately 2mg/kg, and wherein administration of the last initial dose and the firstsubsequent and additional subsequent doses are separated in time by atleast one week.
 56. The article of manufacture of claim 55, wherein theinitial dose is a plurality of doses, wherein each of the plurality ofinitial doses is at least approximately 1 mg/kg and is administered onat least 3 consecutive days, wherein the at least one subsequent dose isat least approximately 6 mg/kg, and wherein administration of the lastinitial dose and the first subsequent and additional subsequent dosesare separated in time by at least 3 weeks.
 57. The article ofmanufacture of claim 38, wherein the initial dose is a plurality ofdoses, wherein each of the plurality of initial doses is at leastapproximately 1 mg/kg and is administered at least twice per week for 3weeks as a first dosage cycle, wherein the dosage cycle is repeated, andwherein the doses of each cycle are separated in time by at least 1 day,and wherein the dosage cycles are separated in time by at least 1 week.58. The article of manufacture of claim 38, wherein the instructions arefor administration of an initial dose by subcutaneous injection and atleast one subsequent dose by subcutaneous injection.
 59. The article ofmanufacture of claim 58, wherein the initial dose is at leastapproximately 6 mg/kg.
 60. The article of manufacture of claim 59,wherein the initial dose is at least approximately 8 mg/kg.
 61. Thearticle of manufacture of claim 60, wherein the initial dose is at leastapproximately 12 mg/kg.
 62. The article of manufacture of claim 58,wherein the subsequent doses are separated from each other in time by atleast one week.
 63. The article of manufacture of claim 58, wherein thesubsequent doses are separated in time from each other by at least twoweeks.
 64. The article of manufacture of claim 58, wherein thesubsequent doses are separated in time from each other by at least threeweeks.
 65. The article of manufacture of claim 58, wherein the initialdose is selected from the group consisting essentially of approximately6 mg/kg, 8 mg/kg, or 12 mg/kg, wherein the plurality of subsequent dosesare at least approximately 2 mg/kg, and wherein the subsequent doses areseparated in time from each other by at least one week.
 66. The articleof manufacture of claim 58, wherein the plurality of subsequent dosesare separated in time from each other by at least two weeks.
 67. Thearticle of manufacture of claim 66, wherein the plurality of subsequentdoses are separated in time from each other by at least three weeks. 68.The article of manufacture of claim 67, wherein the initial dose isapproximately 8 mg/kg, and wherein at least one subsequent dose isapproximately 6 mg/kg.
 69. The article of manufacture of claim 67,wherein the initial dose is approximately 12 mg/kg, and wherein at leastone subsequent dose is approximately 6 mg/kg.
 70. The article ofmanufacture of claim 65, wherein the initial dose is approximately 8mg/kg, and wherein at least one subsequent dose is approximately 8mg/kg.
 71. The article of manufacture of claim 65, wherein the initialdose is approximately 8 mg/kg, wherein at least one subsequent dose is 8mg/kg, and wherein administration of the initial dose and subsequentdoses are separated in time by at least 2 weeks.
 72. The article ofmanufacture of claim 71, wherein the initial dose and subsequent dosesare separated in time by at least 3 weeks.
 73. The article ofmanufacture of claim 58, wherein the initial dose is a plurality ofdoses, wherein each of the plurality of initial doses is at leastapproximately 1 mg/kg and is administered on at least 2 consecutivedays, wherein at least one subsequent dose is at least approximately 2mg/kg, and wherein administration of the last initial dose and the firstsubsequent and additional subsequent doses are separated in time by atleast one week.
 74. The article of manufacture of claim 73, wherein theinitial dose is a plurality of doses, wherein each of the plurality ofinitial doses is at least approximately 1 mg/kg and is administered onat least 3 consecutive days, wherein the at least one subsequent dose isat least approximately 6 mg/kg, and wherein administration of the lastinitial dose and the first subsequent and additional subsequent dosesare separated in time by at least 3 weeks.
 75. The article ofmanufacture of claim 58, wherein the initial dose is a plurality ofdoses, wherein each of the plurality of initial doses is at leastapproximately 1 mg/kg and is administered at least twice per week for 3weeks as a first dosage cycle, wherein the dosage cycle is repeated, andwherein the doses of each cycle are separated in time by at least 1 day,and wherein the dosage cycles are separated in time by at least 1 week.76. The article of manufacture of claim 38, wherein the instructionsfurther include administration of a chemotherapeutic agent.
 77. Thearticle of manufacture of claim 76, wherein the chemotherapeutic agentis a taxoid.
 78. The article of manufacture of claim 77, wherein thetaxoid is paclitaxel or docetaxel.
 79. The article of manufacture ofclaim 76, wherein the chemotherapeutic agent is an anthracyclinederivative.
 80. The article of manufacture of claim 79, wherein theinstructions further include administration of a cardioprotectant. 81.The article of manufacture of claim 38, further comprising a label on orassociated with the container that indicates that said composition canbe used for treating a condition characterized by overexpression ofErbB2 receptor.
 82. The article of manufacture of claim 81, wherein saidlabel indicates that said composition can be used for the treatment ofbreast cancer.
 83. The article of manufacture of claim 38, wherein saidanti-ErbB2 antibody binds to the extracellular domain of the receptor.84. The article of manufacture of claim 83, wherein said anti-ErbB2antibody binds to epitope 4D5 within the ErbB2 extracellular domainsequence.
 85. The article of manufacture of claim 84, wherein saidantibody is a humanized 4D5 anti-ErbB2 antibody.
 86. A method for thetreatment of cancer in a human patient comprising administering to thepatient a first dose of an anti-ErbB2 antibody followed by at least onesubsequent dose of the antibody, wherein the first dose and subsequentdose are separated from each other in time by at least about two weeks.87. The method of claim 86, wherein the first dose and subsequent doseare separated from each other in time by at least about three weeks. 88.The method of claim 86, wherein the first dose and subsequent dose areeach from about 2 mg/kg to about 16 mg/kg.
 89. The method of claim 88,wherein the first dose and subsequent dose are each from about 4 mg/kgto about 12 mg/kg.
 90. The method of claim 89, wherein the first doseand subsequent dose are each from about 6 mg/kg to about 12 mg/kg. 91.The method of claim 86, wherein two or more subsequent doses of theantibody are administered to the patient.
 92. The method of claim 91,wherein from about two to about ten subsequent doses of the antibody areadministered to the patient.
 93. The method of claim 91, wherein the twoor more subsequent doses are separated from each other in time by atleast about two weeks.
 94. The method of claim 93, wherein the two ormore subsequent doses are separated from each other in time by at leastabout three weeks.
 95. The method of claim 91, wherein the two or moresubsequent doses are each from about 2 mg/kg to about 16 mg/kg.
 96. Themethod of claim 91, wherein the two or more subsequent doses are eachfrom about 4 mg/kg to about 12 mg/kg.
 97. The method of claim 91,wherein the two or more subsequent doses are each from about 6 mg/kg toabout 12 mg/kg.
 98. The method of claim 86, wherein the method furthercomprises administering an effective amount of a chemotherapeutic agentto the patient.
 99. The method of claim 98, wherein the chemotherapeuticagent is a taxoid.
 100. An article of manufacture, comprising acontainer, a composition within the container comprising an anti-ErbB2antibody, and a package insert containing instructions to dose theantibody according to any one of claims 86 to
 99. 101. A method for thetreatment of cancer in a human patient, comprising administering aneffective amount of an anti-ErbB antibody to the human patient, themethod comprising: administering to the patient an initial dose of atleast approximately 5 mg/kg of the anti-ErbB antibody; and administeringto the patient a plurality of subsequent doses of the antibody in anamount that is approximately the same or less than the initial dose.102. The method of claim 101 wherein the anti-ErbB antibody is selectedfrom the group consisting of antiepidermal growth factor receptor(EGFR), anti-ErbB3 and anti-ErbB4.
 103. A method for the treatment ofcancer in a human patient comprising administering to the patient afirst dose of an anti-ErbB antibody followed by at least one subsequentdose of the antibody, wherein the first dose and subsequent dose areseparated from each other in time by at least about two weeks.
 104. Themethod of claim 103 wherein the anti-ErbB antibody is selected from thegroup consisting of antiepidermal growth factor receptor (EGFR),anti-ErbB3 and anti-ErbB4.
 105. An article of manufacture, comprising acontainer, a composition within the container comprising an anti-ErbBantibody, and a package insert containing instructions to dose theantibody according to any one of claims 101 to 104.